Compounds, methods, and treatments for abnormal signaling pathways for prenatal and postnatal development

ABSTRACT

The present invention relates to prevention of congenital deformations. The invention further relates to cancer inhibition and prevention. The invention further relates to methods and compositions to modulate, antagonize, or agonize disparate signaling pathways that may converge to regulate patterning events and gene expression during prenatal development, post-natal development, and during development in the adult organism. The invention also relates to activators or deactivators of pyruvate kinase M2 (PKM2) for the treatment, prevention, or amelioration of diseases related to PKM2 function.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 14/487,284, filed Sep. 16, 2014, which is a continuation ofU.S. patent application Ser. No. 13/102,696, filed May 6, 2011, which isa continuation-in-part of U.S. patent application Ser. No. 12/387,239,filed Apr. 30, 2009, now abandoned, which is a continuation-in-part ofU.S. patent application Ser. No. 12/001,869, filed Dec. 13, 2007, nowabandoned, which is a continuation-in-part of U.S. patent applicationSer. No. 11/591,398, filed Nov. 1, 2006, now abandoned, each of which isincorporated by reference in their entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

FIELD OF THE INVENTION

The present invention relates to the field use of inositol stereoisomersand derivatives thereof, especially phosphorylated and carboxylatedderivatives thereof (the invention compounds, some of which are novelcompounds) in a wide range of disease states and medical conditions. Theinvention further relates to use of the invention compounds to modulatesignaling pathways in the development and growth of various cells. Stillfurther, the invention relates to reducing the incidence of fetaldefects due to aberrant pattern formation during gestation. Stillfurther, the invention relates to prevention of fetal alcohol syndromewith invention compounds and combinations. This invention also relatesto novel inhibitors of alpha Tumor Necrosis Factor and protein kinases,pharmaceutical compounds containing inhibitors, and methods forpreparing these inhibitors. They can be useful for the treatment ofhepatitis A, B, C, D, the treatments of chronic inflammation, chronicinflammatory arthritis such as rheumatoid arthritis, other chronicactive diseases, chrohn's disease, inflammatory bowel disease, cancer,autoimmune diseases like lupus, sjogren's, mixed connective tissuedisease, and other pro-inflammatory cytokine-mediated diseases.

The invention still further relates to combination therapy of one ormore of the invention compounds with a substantial range of additionalcompounds, such as anti-cancer therapeutic agents; etc. as furtherdetailed herein (generally as means of reducing tumor load or distantmetastasis, or as a synergistic inhibitor) as well as agents whichprevent or diminish the aberrant cell from obtaining drug resistance;estrogenic or antiandrogenic therapeutic substances (generally as ameans of inhibiting the response of breast tissue to estrogen excessinsult (absolute or relative estrogen excess as compared to normalestrogenic/androgenic substance balance); folic acid or other folatesources (primarily with respect to reducing the incidence of fetalmalformations), etc. as further detailed herein.

Still further, the invention relates to reducing the emergence ofresistance to interferon alpha therapy, to normalizing abnormal activityof TNF as well as kinases active in chronic-active hepatitis (JanusKinase [JAK] and TYK2) activated upon INF-alpha binding, as well asconditions resulting from such abnormal kinase activity. The methods andcompositions can also be used to treat abnormal kinase activity incancers. These include: phosphatidylinositol 3-kinase (PI3K-AKT Mtorsignaling pathway) Ras/MAPK-erk 1/2 pathway, Rapamycin pathway,Insulin-like growth factor(S) signaling, protein kinases includingserine/threonine kinases such as Raf kinases, such as aurora kinases,protein kinase such as MEK, and tyrosine kinases, epidermal growthfactor receptor family (EGFR), platelet-derived growth factor receptorfamily (PDGFR), vascular endothelial growth factor receptor (VEGFR)family, nerve growth factor receptor family (NGFR), fibroblast growthfactor receptor family (FGFR) insulin receptor family, ephrin receptorfamily, Met family, Ror family, c-kit family, Src family, Fes family,Fak family, Syk/ZAP-70 family, Tec family and Abl family. Furthermore,these compounds and methods can be used to upregulate the p27kip1 andP21cip1 and to down regulate or inhibit Ap-1 and ppRb.

More specifically, in some embodiments, the present invention furtherrelates to the phosphatidylinositol/PI3K signaling pathways forprevention or correction of improper signaling in these pathways. Instill other embodiments, the invention relates to compositions for theprevention and/or minimization of fetal malformations, some of which aredue to sonic hedgehog (Shh) and/or other hedgehog variants such asIndian (Ihh) and Desert (Dhh), etc. signaling defects; for theprevention and/or inhibition of breast cancer and metastases thereofsome of which are due to one or more of sequela of estrogen exposure orestrogen surplus exposure (whether during hormonal therapy (males orfemales) or birth control use) or super-active estrogen receptors, ordue to excess number of estrogen receptors (receptor expansion), orexcessively sensitive estrogen receptors in mammary epithelial breasttissue (whether due to derangements in signaling pathways or other basessuch as estrogen receptor overexpression in certain predisposedphenotypes, whether due to developmental, or to environmental, orendogenous exposures). The invention further relates to increasing thetherapeutic efficacy of anti-cancer agents, especially those related tothe prevention or treatment of breast and prostate cancers, and theprevention or reduction of aberrant cells becoming resistant to one ormore anti-cancer agents.

Still further, the invention relates to the Warburg effect onproliferating cells without limitation. The energy that stimulatescancer cells is derived from glycolysis which is the Warburg effect, themetabolic change, the primary alteration in cancer cells which conferssurvival advantage of cancer cells over normal proliferating cells inanimals. One of the most frequently mutated genes in cancer is p53 whichappears to modulate the balance between the utilization of respirationand glycolytic pathways. Cytochrome C oxidaae (SCO2) appears to be thedownstream mediator of this effect. This SCO2 protein enzyme is crucialfor regulating the cytochrome c oxidase (cox complex) which is the majorsite of oxygen use in animals. By disrupting the SCO2 gene orupregulating tumor suppressor gene p53, without being bound to theory,the inventor believes that some of the compounds will turn off themetabolic switch towards glycolysis and deactivate phosphotyrosinebinding or signaling cascades.

The invention also relates to activators or deactivators of pyruvatekinase M2 (PKM2) for the treatment, prevention, or amelioration ofdiseases related to PKM2 function. Pyruvate kinase (PK) is a metabolicenzyme that converts phosphoenolpyruvate to pyruvate during glycolysis.Four PK isoforms exist in mammals: the L and R isoforms are expressed inliver and red blood cells, respectively, the M1 isoform is expressed inmost adult tissues, and the M2 isoform is a splice variant of M1expressed during embryonic development. All tumor cells exclusivelyexpress the embryonic M2 isoform. A well-known difference between the M1and M2 isoforms of PK is that M2 is a low-activity enzyme that relies onallosteric activation by the upstream glycolytic intermediates, whereasM1 is a constitutively active enzyme.

All tumor cells exclusively express the embryonic M2 isoform of pyruvatekinase. PKM2 can serve as a target in cancer therapy. PKM2 is alsoexpressed in adipose tissue and activated T-cells and thus activators ofPKM2 can be used to treat disorders that are dependent on such cells.

While not wishing to be bound by theory, it is believed thatPKM2-dependent cells, e.g., cancer cells, must regulate PKM2, activatingit when the cell's need for completion of glycolysis and maximal ATPproduction is relatively greater and inhibiting it when the cell's needfor anabolic processes (growth) is relatively greater. Thus, theendogenous ability to modulate the activity of PKM2 is criticallyimportant to the cell. Therapeutic or exogenous modulation of PKM2 byinhibition or activation, e.g., constitutive activation or inhibition,defeats the endogenous modulation or regulation by the cell. Activatorscan be used to treat disorders related to PKM2 metabolism, e.g.,disorders characterized by unwanted cell proliferation, e.g., cancer,obesity, diabetes, atherosclerosis, restenosis, and autoimmuneconditions. Selective activators are preferred. Thus, activating PKM2can mean depriving or compromising the ability of a cell to inactivatePKM2. An activator can reduce the cell's ability to down regulate PKM2and can, for example, turn regulated PKM2 activity into constitutivePKM2 activity.

The invention further relates to signaling cascades such as Receptortyrosine kinase/PI3K/AKT/mammalian target of rapamycin (Mtor) onproliferating and tumor cells as an activator of PKM2 or in tumormetabolism and growth. Because of the fact that receptor tyrosinekinase/PI3K/AKT/mammalian target of rapamycin (RTK/PI3K/AKT/mTOR)signaling cascade is a frequently altered pathway in cancer aspreviously described in the parent application. The inventor believesthat receptor tyrosine kinase/PI3K/AKT/mammalian target of rapamycin(RTK/PI3K/AKT/mTOR) may also play a pivotal role as an activator of theWarburg effect by inducing PKM2 and other glycolytic enzymes undernormoxic conditions. By disrupting these pathways that may also have acritical role in cancer cell metabolism, PKM2 upregulation may besuppressed by inhibiting signaling pathways that are addicted to aerobicglycolysis. By dual suppression of signaling pathways, without beingbound to theory, the inventor believes that hyperactive states ofcertain signaling pathways, contributes to the development of theWarburg effect in many cancers. For inhibitingPI3K/mTOR/HIF/MYC-hnRNPS/PKm2 and PKM2 glycolysis signaling network, wehave found utility for the following classes of compounds, including,albeit not limited to any inositol, derivative, or analog. While theD-Chiro inositol analogs are exemplified here, it is understood that allspecies of inositols, their isomeric analogous phosphate derivatives,and/or their phosphatidyl inositol phosphate derivatives are includedherein without limitation.

Tetraphosphates

-   1. D-chiro-inositol 1,2,3,4-tetraphosphate-   2. D-chiro-inositol 1,2,3,5-tetraphosphate-   3. D-chiro-inositol 1,2,3,6-tetraphosphate-   4. D-chiro-inositol 1,2,4,5-tetraphosphate-   5. D-chiro-inositol 1,2,4,6-tetraphosphate-   6. D-chiro-inositol 1,2,5,6-tetraphosphate-   7. D-chiro-inositol 1,3,4,5-tetraphosphate-   8. D-chiro-inositol 1,3,4,6-tetraphosphate-   9. D-chiro-inositol 1,3,5,6-tetraphosphate-   10. D-chiro-inositol 1,4,5,6-tetraphosphate-   11. D-chiro-inositol 2,3,4,5-tetraphosphate-   12. D-chiro-inositol 2,3,4,6-tetraphosphate-   13. D-chiro-inositol 2,3,5,6-tetraphosphate-   14. D-chiro-inositol 2,4,5,6-tetraphosphate-   15. D-chiro-inositol 3,4,5,6-tetraphosphate    Pentaphosphates-   1. D-chiro-inositol 1,2,3,4,5-pentaphosphate-   2. D-chiro-inositol 1,2,3,4,6-pentaphosphate-   4. D-chiro-inositol 1,3,4,5,6-pentaphosphate-   5. D-chiro-inositol 1,3,4,5,6-pentaphosphate-   6. D-chiro-inositol 1,3,4,5,6-pentaphosphate    Hexaphosphates-   1. D-chiro-inositol 1,2,3,4,5,6-hexaphosphate    Heptaphosphates-   1. D-chiro-inositol 1,2,3,4,5-pentaphosphate-6-pyrophosphate-   2. D-chiro-inositol 1,2,3,4,6-pentaphosphate-5-pyrophosphate-   3. D-chiro-inositol 1,2,3,5,6-pentaphosphate-4-pyrophosphate-   4. D-chiro-inositol 1,2,4,5,6-pentaphosphate-3-pyrophosphate-   5. D-chiro-inositol 1,3,4,5,6-pentaphosphate-2-pyrophosphate-   6. D-chiro-inositol 2,3,4,5,6-pentaphosphate-1-pyrophosphate-   7. D-chiro-inositol 1,2,3-triphosphate-4,5-dipyrophosphate-   8. D-chiro-inositol 1,2,3-triphosphate-4,6-dipyrophosphate-   9. D-chiro-inositol 1,2,3-triphosphate-5,6-dipyrophosphate-   10. D-chiro-inositol 1,2,4-triphosphate-3,5-dipyrophosphate-   11. D-chiro-inositol 1,2,4-triphosphate-3,6-dipyrophosphate-   12. D-chiro-inositol 1,2,4-triphosphate-5,6-dipyrophosphate-   13. D-chiro-inositol 1,2,5-triphosphate-3,4-dipyrophosphate-   14. D-chiro-inositol 1,2,5-triphosphate-3,6-dipyrophosphate-   15. D-chiro-inositol 1,2,5-triphosphate-4,6-dipyrophosphate-   16. D-chiro-inositol 1,2,6-triphosphate-3,4-dipyrophosphate-   17. D-chiro-inositol 1,2,6-triphosphate-3,5-dipyrophosphate-   18. D-chiro-inositol 1,2,6-triphosphate-4,6-dipyrophosphate-   19. D-chiro-inositol 1,3,4-triphosphate-2,5-dipyrophosphate-   20. D-chiro-inositol 1,3,4-triphosphate-2,6-dipyrophosphate-   21. D-chiro-inositol 1,3,4-triphosphate-5,6-dipyrophosphate-   22. D-chiro-inositol 1,3,5-triphosphate-2,4-dipyrophosphate-   23. D-chiro-inositol 1,3,5-triphosphate-2,6-dipyrophosphate-   24. D-chiro-inositol 1,3,5-triphosphate-4,6-dipyrophosphate-   25. D-chiro-inositol 1,3,6-triphosphate-2,4-dipyrophosphate-   26. D-chiro-inositol 1,3,6-triphosphate-2,5-dipyrophosphate-   27. D-chiro-inositol 1,3,6-triphosphate-4,5-dipyrophosphate-   28. D-chiro-inositol 1,4,5-triphosphate-2,3-dipyrophosphate-   29. D-chiro-inositol 1,4,5-triphosphate-2,6-dipyrophosphate-   30. D-chiro-inositol 1,4,5-triphosphate-3,6-dipyrophosphate-   31. D-chiro-inositol 1,4,6-triphosphate-2,3-dipyrophosphate-   32. D-chiro-inositol 1,4,6-triphosphate-2,5-dipyrophosphate-   33. D-chiro-inositol 1,4,6-triphosphate-3,5-dipyrophosphate-   34. D-chiro-inositol 1,5,6-triphosphate-2,3-dipyrophosphate-   35. D-chiro-inositol 1,5,6-triphosphate-2,4-dipyrophosphate-   36. D-chiro-inositol 1,5,6-triphosphate-3,4-dipyrophosphate-   37. D-chiro-inositol 2,3,4-triphosphate-1,5-dipyrophosphate-   38. D-chiro-inositol 2,3,4-triphosphate-1,6-dipyrophosphate-   39. D-chiro-inositol 2,3,4-triphosphate-5,6-dipyrophosphate-   40. D-chiro-inositol 2,3,5-triphosphate-1,4-dipyrophosphate-   41. D-chiro-inositol 2,3,5-triphosphate-1,6-dipyrophosphate-   42. D-chiro-inositol 2,3,5-triphosphate-4,6-dipyrophosphate-   43. D-chiro-inositol 2,3,6-triphosphate-1,4-dipyrophosphate-   44. D-chiro-inositol 2,3,6-triphosphate-1,5-dipyrophosphate-   45. D-chiro-inositol 2,3,6-triphosphate-4,5-dipyrophosphate-   46. D-chiro-inositol 2,4,5-triphosphate 1,3-dipyrophosphate-   47. D-chiro-inositol 2,4,5-triphosphate-1,6-dipyrophosphate-   48. D-chiro-inositol 2,4,5-triphosphate-3,6-dipyrophosphate-   49. D-chiro-inositol 2,4,6-triphosphate-1,3-dipyrophosphate-   50. D-chiro-inositol 2,4,6-triphosphate-1,5-dipyrophosphate-   51. D-chiro-inositol 2,4,6-triphosphate-3,5-dipyrophosphate-   52. D-chiro-inositol 2,5,6-triphosphate-1,3-dipyrophosphate-   53. D-chiro-inositol 2,5,6-triphosphate-1,4-dipyrophosphate-   54. D-chiro-inositol 2,5,6-triphosphate-3,4-dipyrophosphate-   55. D-chiro-inositol 3,4,5-triphosphate-1,2-dipyrophosphate-   56. D-chiro-inositol 3,4,5-triphosphate-1,6-dipyrophosphate-   57. D-chiro-inositol 3,4,5-triphosphate-2,6-dipyrophosphate-   58. D-chiro-inositol 3,5,6-triphosphate-1,2-dipyrophosphate-   59. D-chiro-inositol 3,5,6-triphosphate-1,4-dipyrophosphate-   60. D-chiro-inositol 3,5,6-triphosphate-2,4-dipyrophosphate-   61. D-chiro-inositol 4,5,6-triphosphate-1,2-dipyrophosphate-   62. D-chiro-inositol 4,5,6-triphosphate-1,3-dipyrophosphate-   63. D-chiro-inositol 4,5,6-triphosphate-2,3-dipyrophosphate-   64. D-chiro-inositol 1-phosphate-2,3,4-tripyrophosphate-   65. D-chiro-inositol 1-phosphate-2,3,5-tripyrophosphate-   66. D-chiro-inositol 1-phosphate-2,3,6-tripyrophosphate-   67. D-chiro-inositol 1-phosphate-2,4,5-tripyrophosphate-   68. D-chiro-inositol 1-phosphate-2,4,6-tripyrophosphate-   69. D-chiro-inositol 1-phosphate-2,5,6-tripyrophosphate-   70. D-chiro-inositol 1-phosphate-3,4,5-tripyrophosphate-   71. D-chiro-inositol 1-phosphate-3,4,6-tripyrophosphate-   73. D-chiro-inositol 1-phosphate-4,5,6-tripyrophosphate-   74. D-chiro-inositol 2-phosphate-1,3,4-tripyrophosphate-   76. D-chiro-inositol 2-phosphate-1,3,6-tripyrophosphate-   77. D-chiro-inositol 2-phosphate-1,4,5-tripyrophosphate-   78. D-chiro-inositol 2-phosphate-1,4,6-tripyrophosphate-   79. D-chiro-inositol 2-phosphate-1,5,6-tripyrophosphate-   80. D-chiro-inositol 2-phosphate-3,4,5-tripyrophosphate-   81. D-chiro-inositol 2-phosphate-3,4,6-tripyrophosphate-   82. D-chiro-inositol 2-phosphate-3,5,6-tripyrophosphate-   83. D-chiro-inositol 2-phosphate-4,5,6-tripyrophosphate-   84. D-chiro-inositol 3-phosphate-1,2,4-tripyrophosphate-   85. D-chiro-inositol 3-phosphate-1,2,5-tripyrophosphate-   86. D-chiro-inositol 3-phosphate-1,2,6-tripyrophosphate-   87. D-chiro-inositol 3-phosphate-1,4,5-tripyrophosphate-   88. D-chiro-inositol 3-phosphate-1,4,6-tripyrophosphate-   89. D-chiro-inositol 3-phosphate-1,5,6-tripyrophosphate-   90. D-chiro-inositol 3-phosphate-3,4,5-tripyrophosphate-   91. D-chiro-inositol 3-phosphate-3,4,6-tripyrophosphate-   92. D-chiro-inositol 3-phosphate-3,5,6-tripyrophosphate-   93. D-chiro-inositol 3-phosphate-4,5,6-tripyrophosphate    Other Derivatives of Inositol as PI3K/AKT/Inhibitors-   1.    D-1-deoxy-2-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   2.    D-1-deoxy-3-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   3.    D-1-deoxy-4-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   4.    D-1-deoxy-5-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   5.    D-1-deoxy-6-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   6.    D-2-deoxy-1-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   7.    D-2-deoxy-3-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   8.    D-2-deoxy-4-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   9.    D-2-deoxy-5-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   10.    D-2-deoxy-6-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   11. D-Chiro phosphatidyl inositol    D-3-deoxy-1-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   12. D-Chiro phopshatidyl inositol    D-3-deoxy-2-O-methyl-D-chiroinositol-8    [1-(R)-2-methoxy-3-(octadecyloxy)propyl hydrogen phosphate]-   13. D-Chiro phopshatidyl inositol    D-3-deoxy-4-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   14. D-Chiro phopshatidyl inositol    D-3-deoxy-5-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   15. D-chiro phopshatidyl inositol    D-3-deoxy-6-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   16.    D-4-deoxy-1-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   17.    D-4-deoxy-2-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   18.    D-4-deoxy-3-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   19.    D-4-deoxy-3-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   20.    D-4-deoxy-6-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   21.    D-5-deoxy-1-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   22.    D-5-deoxy-2-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   23.    D-5-deoxy-3-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   24.    D-5-deoxy-4-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   25.    D-5-deoxy-6-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   26.    D-6-deoxy-1-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   27.    D-6-deoxy-2-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   28.    D-6-deoxy-3-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   29.    D-6-deoxy-4-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   30.    D-6-deoxy-5-O-methyl-D-chiroinositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl    hydrogen phosphate]-   1.    1D-6-hydroxymethyl-chiro-inositol-1-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   2.    1D-6-hydroxymethyl-chiro-inositol-2-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   3.    1D-6-hydroxymethyl-chiro-inositol-3-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   4.    1D-6-hydroxymethyl-chiro-inositol-4-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   5.    1D-6-hydroxymethyl-chiro-inositol-5-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   6.    1D-5-hydroxymethyl-chiro-inositol-1-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   7.    1D-5-hydroxymethyl-chiro-inositol-2-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   8.    1D-5-hydroxymethyl-chiro-inositol-3-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   9.    1D-5-hydroxymethyl-chiro-inositol-4-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   10.    1D-5-hydroxymethyl-chiro-inositol-6-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   11.    1D-4-hydroxymethyl-chiro-inositol-1-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   12.    1D-4-hydroxymethyl-chiro-inositol-2-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   13.    1D-4-hydroxymethyl-chiro-inositol-3-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   14.    1D-4-hydroxymethyl-chiro-inositol-5-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   15.    1D-5-hydroxymethyl-chiro-inositol-6-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   16.    1D-3-hydroxymethyl-chiro-inositol-1-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   17.    1D-3-hydroxymethyl-chiro-inositol-2-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   18.    1D-3-hydroxymethyl-chiro-inositol-4-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   19.    1D-3-hydroxymethyl-chiro-inositol-5-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   20.    1D-3-hydroxymethyl-chiro-inositol-6-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   21.    1D-2-hydroxymethyl-chiro-inositol-1-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   22.    1D-2-hydroxymethyl-chiro-inositol-3-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   23.    1D-2-hydroxymethyl-chiro-inositol-4-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   24.    1D-2-hydroxymethyl-chiro-inositol-5-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   25.    1D-2-hydroxymethyl-chiro-inositol-6-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   26.    1D-1-hydroxymethyl-chiro-inositol-2-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   27.    1D-1-hydroxymethyl-chiro-inositol-3-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   28.    1D-1-hydroxymethyl-chiro-inositol-4-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   29.    1D-1-hydroxymethyl-chiro-inositol-5-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate-   30.    1D-1-hydroxymethyl-chiro-inositol-6-(R)-2-methoxy-3-O-octadecyl-sn-glycerocarbonate

Another aspect of the invention is to regulate metabolic pathways inrapidly growing tissues. This relates to all cancer cells, obesity,diabetes, neurodegenerative diseases, and inflammation withoutlimitation to inflammatory conditions or autoimmune. The inventions alsoshow methods, compositions, and kits for the use of activators ordeactivators of PKM2 or PKM1 for increasing tumor suppressor genes,specifically P53 or deactivating a wild type p53 (defective tumorsuppressor gene).

Another aspect of the invention is to activate or upregulate P53 tumorsuppressor with inositol and or phosphates (but not limited in any way).P53 is one of the most frequently mutated genes in cancer. P53 appearsto modulate the balance between the utilization of respiration andglycolytic pathways and Cytochrome C oxidase (SCO2) appears to be thedownstream mediator of this effect. This SCO2 protein enzyme is crucialfor regulating the cytochrome c oxidase (cox complex) which is the majorsite of oxygen use in animals. By disrupting the SCO2 gene orupregulating tumor suppressor gene p53 with inositol phosphates, theinventor believes that the compounds will turn off the metabolic switchtowards glycolysis and deactivate phosphotyrosine binding or signaling.

In yet other aspects of the invention, it relates to manipulating cellgrowth and differentiation in culture for implantation of such cells. Inthis context, the invention relates to treatments for regeneratingneural tissue, hepatic tissue, pancreatic tissue, intestinal tissue,spleenic tissue, cardiac tissue, among others. In still otherembodiments, the invention relates to regulating or inhibiting growth ofcells and therefore finds use in the treatment of excessive orinappropriate hair growth conditions, psoriasis, actinic keratosis,acne, miscellaneous dermatitis conditions, etc. In yet otherembodiments, the invention relates to inducing an anti-angiogenic statein local and distant metastatic tumors.

The invention further relates to correcting the inherent mechanism oftumor stem cell autoregulation. Yet another embodiment of the inventionrelates to decreasing the risk of deep vein thrombosis (DVT's) andPulmonary emboli (PE's) while using chemotherapeutic agents,antiestrogens such as tamoxifen etc., hormonal therapies such asandrogen ablative therapies, or estrogenic hormone therapy. Theinvention still further relates to reducing the numbers and size oftumors locally or distant especially in breast cancers, but also cancersoriginating from blood, colon, lung, liver, pancreas, cervix, prostate,skin, and soft tissue.

The invention further relates to preventing breast cancer or precursorsthereof in utero.

BACKGROUND OF THE INVENTION

The present invention relates to compounds that are of the inositolfamily and for many of the uses set forth herein specifically theD-chiroinositol family of compounds. Most are derivatives of the basicparent of the family, but in some indications, even the basic parent wasnot previously known or thought to be useful in the contexts indicated.As indicated in the above fields of the invention, the invention isapplicable to a wide array of utilities a number of which are addressedseparately in this background section below.

I. Fetal Malformation

Fetal malformations are a continuing medical problem in serious need ofprevention and treatment. These malformations can result in innocuousdefects that pose no health or psychological issues, to those that poseprimarily social or psychological issues (such as webbed digits, etc.),to those that pose medical issues of varying degrees of severity. Someof the more medically severe malformations include neural tube defects(such as, among others, anencephaly where the brain is underdeveloped orthere is an incomplete skull, encephalocele, where there is a hole inthe skull through which tissue protrudes, and spina bifida, where aportion of the spine is exposed) to cranio-facial defects (such as,among others, cleft lip and cleft palate) to imperforate anus (where theanal opening doesn't form properly leaving no exit for intestinalcontents, or intestinal/rectal emptying into inappropriate structuressuch as the bladder, ureter, uterus or vagina). Other birth defects likeneural derived brain tumors are not so evident immediately after birthand rarely if ever seen at birth. For example, the onset of pediatrictumors like primitive neuroectodermal tumors of the central nervoussystem (PNET'S) is insidious. There is considerable controversyregarding the histiogenesis of these tumors, but a genetic loci ofinterest in the pathnogenesis of these central nervous system derivedtumors includes Shh gene pathways.

Cleft palate children were found to have low red blood cell zinc levelsand low myo-inositol levels (Krapels, et al: Myo-inositol, glucose andzinc status as risk factors for non-syndromic cleft lip with or withoutcleft palate in offspring: a case-control study, BJOG. 2004 July;111(7):661-8). Impairment of the Folbp1 gene function adversely impactsthe expression of several critical signaling molecules. Mis-expressionof these molecules, perhaps mediated by Shh may potentially contributeto the observed failure of the neural tube and the development ofcraniofacial defects in the mutant mice (Birth Defects Research, WileyInterscience, 2003).

II. Signaling Pathways

In molecular biology, ‘signal transduction’ refers to any process bywhich a cell converts one kind of signal or stimulus into another. Mostprocesses of signal transduction involve ordered sequences ofbiochemical reactions inside the cell, which are carried out by enzymes,activated by second messengers, resulting in the signal transductionpathway. These processes are usually quick, lasting milliseconds in thecase of sodium, potassium and calcium ion fluxes, or minutes for theactivation of protein and lipid-mediated kinase cascades. However, somesignaling cascade events can take days and many hours. (As is the casewith gene expression), to complete. The number of proteins and othermolecules participating in the events involving signal transductionincreases as the process starts from the initial stimulus, resulting ina “signal cascade,” that usual begins with a relatively small stimulusthat elicits a larger response. This is referred to as “amplification ofthe signal” where the signal spreads across a spatial concentrationgradient. The continued cell growth and/or death of a cell are some ofthe cellular responses to extracellular stimulation that require signaltransduction as well. Gene activation leads to further cellular effects,like activation of transcription factors which are also the result ofanother signal transduction cascade. This cascade leads to activate yetmore genes.

Most mammalian cells require constant growth factor stimulation tocontrol not only cell division but also survival. In the absence ofsignal transduction, programmed cell death ensues in most cells. Suchrequirements for extra-cellular stimulation are necessary forcontrolling cell behavior in the multi-cellular organisms. Signaltransduction pathways are known to be so central to biological processesthat it is not surprising that a large number of diseases have beenattributed to their disregulation and cross talk. Discussed below is howsignal transduction can disrupt genes that can lead to various diseases.

Disruptions of genes in one pathway can also have deleterious effects inother pathways and may result in serious dysmorphogensis or cancer yearslater. For a better understanding of this aspect of the presentinvention, a basic overview of some of the genes and signaling pathwaysthat may be affected by consequences of alterations in genes, theirproducts, and specific exposures is in order. This also requires someunderstanding of the pathways involved in formation, embryonicdevelopment, and cancer.

During embryogenesis a simple, patterned body plan is established. Toestablish this organization, the cells of the embryo need to becomespecified and must differentiate into cell types in an integratedmanner. The genetic regulation of this process is addressed here. Inanimals, cell-cell communication involving extracellular signals andcell surface-bound receptors plays an important role in cell fatedecisions during embryogenesis (Johnston and Nusslein-Volhard, 1992).Probably the best characterized signaling events in embryo patterningare those that involve sonic hedgehog (Shh). Mutations in the Shh genesand the genes that encode its downstream intracellular signaling pathwayand subsequent responses can set the patter for various birth defectsand cancers in both prenatal and post natal development (Mol Med Today,1998). Pattern formation is the activity by which embryonic cells formordered spatial arrangements of differentiated tissues. The physicalcomplexity of higher organisms arises during embryogenesis through theinterplay of cell-intrinsic lineage and cell-extrinsic signaling.Inductive interactions are involved in embryonic patterning invertebrate development from the earliest establishment of the body plan,to the patterning of the organ systems, to the generation of diversecell types during tissue differentiation (Davidson, E., (1990)Development 108: 365-389; Gurdon, J. B., (1992) Cell 68: 185-199;Jessell, T. M. et al., (1992) Cell 68: 257-270). The effects ofdevelopmental cell interactions are varied. Typically, responding cellsare diverted from one route of cell differentiation to another byinducing cells that differ from both the uninduced and induced states ofthe responding cells (inductions). Sometimes cells induce theirneighbors to differentiate like themselves (homeogenetic induction); inother cases a cell inhibits its neighbors from differentiating likeitself. Cell interactions in early development may be sequential orconvergent, such that an initial induction between two cell types leadsto a progressive amplification of diversity. Moreover, inductiveinteractions occur not only in embryos, but in adult cells as well, andcan act to establish and maintain morphogenetic patterns as well asinduce differentiation in cell types (J. B. Gurdon (1992) Cell68:185-199). These cells can also act to establish and maintainmorphogenetic patterns as well as induce differentiation (J. B. Gurdon(1992) Cell 68:185-199). The involvement of signal transduction pathwaysin the inhibition of gene expression and the occurrence of frequentallelic deletions in humans and animals support a tumor suppressorfunction for these pathways. Their role in the regulation of embryonicpattern formation known to be involved in cell signaling andintercellular communication provides a possible mechanism of fetalmalformations, birth defects, autoimmune inflammatory diseases,inflammation, drug resistance, anti-angiogenesis, and cancers.

Notwithstanding the above, there is still a tremendous amount that isstill not fully understood in the art about the nature of the all themechanisms involved in the etiology of these malformations and diseasesand how to appropriately intervene to reduce or prevent the occurrenceof such defects. Thus, we are also investigating the possibility thatmany signaling pathways comprise multiple steps and cross convergencesalong the feedback loop.

Nevertheless, we are closer to prevention of these diseases, and bettertreatment options that will eventually improve the quality of manypatients' lives. The role of D-chiro-inositol its steroisomers and/orits phosphates and other derivatives among these pathways mentionedabove, and their likely cross-talk in regulation of embryonicpatterning, cancer, inflammatory autoimmune diseases, inflammation isnow addressed in this application.

III. Fetal Alcohol Syndrome

Fetal alcohol syndrome (FAS) is one of the most common, preventablecauses of devastating congenital structural birth defects to majororgans like the brain, heart and kidneys and to varying defects of theface and limbs. It may occur as often as neural tube defects. Accordingto recent literature, at least 33% to 50% of children born to chronicalcoholic mothers show some signs of fetal alcohol syndrome. Fetalalcohol syndrome remains a significant psychosocial and clinicalchallenge. While there have been great strides in preventing neural tubedefects with folic acid, there are no known truly preventativestrategies for preventing fetal alcohol syndrome related birth defects.One recent article (Li, et al; Fetal alcohol exposure impairs hedgehogcholesterol modification and signaling, Laboratory Investigation (2007)87, 231-240 (Jan. 22, 2007)) discusses results in the zebrafish thatexposure of zebrafish embryos to low levels of alcohol duringgastrulation blocks modification of Sonic hedgehog by cholesterol andlead to permanent developmental defects that resemble fetal alcoholsyndrome defects, and further that administration of cholesterol rescuesthe Sonic hedgehog function and prevents the defects in zebrafish.Unfortunately, cholesterol has a significant number of problems of itsown that make it generally an unacceptable therapy for use in the humanpopulation and other treatment avenues are desired.

Ofori, et al Risk of congenital anomalies in pregnant users of statindrugs, British Journal of Clinical Pharmacology, vol. 64, No. 4, October2007, pp 496-509, states that because cholesterol is known to beessential for fetal development, statins, which inhibit cholesterolproduction, have been considered potential teratogens, and thereforehave been contraindicated in pregnancy. Ofori states that from theirdatabase of 288 they did not find evidence of increased risk in womenwho filled prescriptions for statins during pregnancy as compared tothose who stopped statin use before pregnancy (between 1 year and 1month before pregnancy) and compared to women who used fibrates duringpregnancy based on live births. They do state however, that conclusionsremain uncertain due to the lack of data about non-live births. Therewere no matched controls in this study either.

A posting on www.medpagetoday.com/OBGYN/Pregnancy/11317 published Oct.14, 2008, by Gever, et al Statins May Prevent Some Miscarriagesindicates that antiphospholipid antibody seen with patients withautoimmune inflammatory diseases induced miscarriages may be preventedby administration of the statins simvastatin and pravastatin. Thus,statin use in pregnancy in those showing predisposition to or symptomsof antiphospholipid antibodies is desirable. (See also Weiler, Tracingthe molecular pathogenesis of antiphospholipid syndrome, J. Clin.Invest. 118(10): 3276-3278 (2008)).

IV. Autoimmune and Inflammatory Diseases

The present invention also relates to inflammatory autoimmune diseasesthat are triggered and promoted by abnormal kinase activity due todefective cell signaling. Interferon alpha (IFN-alpha) has been used fora decade to treat viral hepatitis, is a disease that effectsapproximately 350 million per year worldwide, more than half of thoseviral hepatitis patients respond poorly to drugs. It appears that highlevels of tumor necrosis factor (TNF) correlate highly with resistanceto interferon alpha therapy. There is an urgent need to find appropriatetreatments for interferon resistance without adding more side effects tothe already compromised liver. The methods and compounds are providedfor treating diseases associated with abnormal activity of proteinkinases. The method comprises administration of a specific or selectiveAKT inhibitor (for exampleD-3-Deoxy-2-O-methyl-D-Chiro-inositol-[1-(R)-2-methoxy-3-(octadecyloxy)propyl HPO4-](hydrogen phosphate)) with interferon alpha (IFN-alpha) toa patient in therapeutically effective amounts; and administering akinase inhibitor to the patient in therapeutically effective amount,such that the in viva activity of the high levels of tumor necrosisfactor (TNF) is reduced relative to that prior to the treatment so inorder to overcome resistance to INF-alpha therapy. Interferon-alpha iscurrently the only well-established treatment for chronic-active viralhepatitis that affects millions world wide. It is thought that theactivity of interferon is mediated by inducing expression of antiviralproteins and modulation of the immune system. Even though interferon hasbeen used widely for many years for the treatment of viral hepatitis,most of these patients respond poorly. The mechanism underlying thisproblem remains unclear at this point, although both genetic andmolecular implications must be considered. There have been many attemptsto improve patient response to interferon by combining interferon withother anti-viral agents (i.e., Ribivarin). It appears that only a smallnumber of patients responded to this attempt to increase the efficacy ofcombined interferon and Ribivarin. Thus, it is an urgent medical need todevelop a new combination of compounds to improve interferon alpharesponse in patients with chronic-active hepatitis.

A number of acute and chronic inflammatory diseases have also beenassociated with very high amounts of circulating inflammatory cytokinesand interleukin 1,6,8. These cytokines include but not limited to Tumornecrosis factor (TNF)-a cytokine involved in systemic inflammation. TNFalso causes apoptotic cell death, cellular proliferation, inflammation,differentiation, tumorigenesis, and viral replication. But TNF's primaryrole is in the regulation of immune type of cells and theirdysregulation and overproduction have been implicated in those diseasesdescribed above. Interleukin 1 beta, interleukin 6, interleukin8 arealso other types of cytokines responsible for inflammatory response in avariety of human diseases.

The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway plays apivotal role in cellular proliferation and growth signaling as describedabove. It involves the downstream activation of the protein kinase Akt.PtdIns (3,4) P2(biphosphate) and ptdIns(3,4,5)P3(triphosphate) lipidsproduced by PI3K are able to bind Akt recruiting it to the plasmamembrane where PDK1 and PDK2 phosphorylate it to its active form. Thisallows Akt to target proteins involved in cell death. Due to theconstitutive activation of PI3K pathway in many cancers and immunemediated signaling pathways this pathway may produce systemicautoimmunity and resistance to interferon alpha for people with chronic,active viral hepatitis and chronic autoimmune inflammatory disease.Therefore, the inventor's research is helping to elucidate the role ofthis pathway and how perturbations that leads to a disregulatedactivation in this pathway. Inhibition of this pathway with combinationtherapy is a novel treatment for cancer and inflammatory disease states.

The phosphatidylinositol 3-[OH] kinase (PI3K)/Akt-(Protein kinase B)pathway is also important for survival such as platelet derived growthfactor (PDGF), nerve growth factor (NGF) and insulin-like growthfactor-1 (IGF-1), promote cell survival under various conditions byinducing the activity of PI3K. Activated PI3K leads to the production ofphosphatidylinositol (3,4,5)-triphosphate (PtdIns(3,4,5)-P3), a secondmessenger which in turn binds to, and promotes the activation of, theserine/threonine kinase Akt, which contains a pleckstrin homology(PH)-domain (Franke et al, Cell, 88:435-437, 1997).

Recent Literature analysis of AKT levels in autoimmune inflammatorydiseases protein Kinase B (Akt) appears to be overexpressed orhyperactive in a significant number of inflammatory autoimmune diseasessuch as Rheumatoid arthritis, Systemic lupus, Inflammatory BowelDiseases, Chron's disease and others. It is also postulated here thathigh levels of tumor necosis factor is mediated by dyregulated PI3ksignaling which can lead to Inteferon alpha resistance seen in manypatients with viral hepatitis. Furthermore, PTEN is a tumor suppressorgene mutated in many human sporadic cancers and hyperactive immunesystems. Those with hereditary cancer syndromes such as Bannayan-Zonanasyndrome and Cowden syndromes are purportedly pten deficient (Lieaw etal. nature genetics 16:64-67. Functionally, PTEN is a dual protein andlipid phosphatase enzyme (Li et al. science 275:1943-1947, 1997) and themajor substrate of PTEN is phosphatidyl inositol-3,4,5-triphosphate(PIP3), a second messenger molecule then produced through PI3K signalingactivation induced by growth factor stimulation. PIP3 activates theserine-threonine kinase PKB/Akt which is involved in anti-apoptosis,proliferation, and oncogenesis. PTEN negatively regulates cell survivalby dephosphorylating PIP3 by enzymatic activation. Recent literaturecited studies that showed that a null mutation of Pten in mice is lethalduring embryogenesis. In a study, it was demonstrated that PKB/Aktpathway is hyperactivated in the absence of Pten). Furthermore,Pten^(+/−) mice frequently develop T cell lymphomas, and endometrial,prostatic, and breast cancers. Autoimmune disorders are also prevalentin Pten deficient mice. In T cell-specific Pten-deficient mice, itshowed that CD4⁺ lymphomas and autoimmune disorders were prevalent.These observations support the role of PI3K/Akt signaling pathways playan important role for regulating cell survival, apoptosis intransforming cells into cancerous, metastatic tumors.

Three members of the Akt/PKB subfamily of second-messenger regulatedserine/threonine protein kinases have been identified and termedAkt1/PKB.alpha., Akt2/PKB.beta., and Akt3/PKB.gamma., respectively. Theisoforms are homologous, particularly in regions encoding the catalyticdomains. Akt/PKBs are activated by phosphorylation events occurring inresponse to PI3K signaling. PI3K phosphorylates membrane inositolphospholipids, generating the second messengers phosphatidyl-inositol3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate, whichhave been shown to bind to the PH domain of Akt/PKB. The current modelof Akt/PKB activation proposes recruitment of the enzyme to the membraneby 3′-phosphorylated phosphoinositides, where phosphorylation of theregulatory sites of Akt/PKB by the upstream kinases occurs (B. A.Hemmings, Science 275:628-630 (1997); B. A. Hemmings, Science 276:534(1997); J. Downward, Science 279:673-674 (1998)).

Phosphorylation of Akt1/PKB.alpha occurs on two regulatory sites,Thr.sup.308 in the catalytic domain activation loop and on Ser.sup.473near the carboxy terminus (D. R. Alessi et al. EMBO J. 15:6541-6551(1996) and R. Meier et al. J. Biol. Chem. 272:30491-30497 (1997).Equivalent regulatory phosphorylation sites occur in Akt2/PKB.beta andAkt3/PKB.gamma. The upstream kinase, which phosphorylates Akt/PKB at theactivation loop site has been cloned and termed 3′-phosphoinositidedependent protein kinase 1 (PDK1). PDK1 phosphorylates not only Akt/PKB,but also p70 ribosomal S6 kinase, p90RSK, serum andglucocorticoid-regulated kinase (SGK), and protein kinase C (B. A.Hemmings, Science 276:534 (1997); J. Downward, Science 279:673-674(1998).

Inhibition of Akt activation and activity can be achieved by inhibitingPI3K with inhibitors already known in the prior art such as LY294002 andwortmannin. However, some research has suggested PI3K inhibition has thepotential to indiscriminately affect not just all three Akt isozymes butalso other PH domain-containing signaling molecules that are dependenton PtdIns(3,4,5)-P3, such as the Tee family of tyrosine kinases.Furthermore, it has been disclosed that Akt can be activated by growthsignals that are independent of PI3K.

At the same time, Akt activity can be inhibited by blocking the activityof the upstream kinase PDK1. No specific PDK1 inhibitors have beendisclosed. However, a recent study interestingly suggested thatIns(1,3,4,5,6)P₅ and InsP₆, as well as Ins(1,3,4,5)P₄, were found tobind PDK1 with high affinity, with K_(i) values of 20-60 nM. Incontrast, Ins (1,4,5)P₃ binding to PDK1 could not be detected. Thehydrogen bonds to the D1-phosphate appear to play a crucial role inmediating binding of inositol phosphates to PDK1. Furthermore, they nextinvestigated whether Ins(1,3,4,5,6)P₅ and InsP₆ could compete withPtdIns(3,4,5)P₃ in regulating the activation of PKB by PDK1 in an invitro assay. The results showed with a dose given of 300 μM InsP₆ orIns(1,3,4,5,6)P₅ inhibited the activation and phosphorylation of PKB inthe presence of PtdIns(3,4,5)P₃, by over 80%. InsP₆ or Ins(1,3,4,5,6)P₅did not directly inhibit PDK1 catalytic activity, as the phosphorylationof the PDKtide peptide substrate by PDK1 was not affected byD6-phosphorylated inositol phosphates. It has also been reported thatincreasing extracellular Ins(1,3,4,5,6)P₅ in cells lowered PKBactivation, through an undefined mechanism (Piccolo et al, 2004). It isthe inventor's belief that different inositol polyphosphates interactdifferently. Some agozine the activity in the presence of othersubstrates while others antagonize the activation of these signalingpathways. EMBO J. 2004 October 13; 23(20): 3918-3928.

The inventor does not believe that the inhibition of PDK1 would resultin inhibition of multiple protein kinases whose activities depend onPDK1, such as atypical Protein Kinase C PKC isoforms, SGK, and S6kinases if the different invention compounds were utilized in aselective, specific manner in the presence of different substrates. Forexample, phosphorylation of PKC by PDK-1 may not require the PH domainof PDK-1 and may not be affected by 3′-phosphoinositides. Thus, despitewhat earlier literature suggests, the inventor believes that perhaps notall of these enzymes are dependent upon phosphoinositide phosphorylationand that there may be some other lipid signaling pathway present that isinvolved in cross-talk.

Therefore, further embodiments of the invention compounds are envisionedto be selective for inhibiting different isoenzymes. The compounds ofthe instant invention are inhibitors of the activity of Akt and PDK1 arethus useful in the treatment of cancer, in particular cancers associatedwith irregularities in the activity of Akt and/or GSK3. Such cancersinclude, but are not limited to ovarian, pancreatic and breast cancer.

In an embodiment of the invention, the instant compound is a selectiveinhibitor whose inhibitory efficacy is dependent on the PH domain. Inthis embodiment, the compound exhibits a decrease in in vitro inhibitoryactivity or no in vitro inhibitory activity against truncated Aktproteins lacking the PH domain.

In a further embodiment, the instant compound is selected from the groupof a selective inhibitor of Akt1, a selective inhibitor of Akt2 and aselective inhibitor of both Akt1 and Akt2.

In another embodiment, the instant compound is selected from the groupof a selective inhibitor of Akt1, a selective inhibitor of Akt2, aselective inhibitor of Akt3 and a selective inhibitor of two of thethree Akt isoforms.

In another embodiment, the instant compound is a selective inhibitor ofall three Akt isoforms, but is not an inhibitor of one, two or all ofsuch Akt isoforms that have been modified to delete the PH domain, thehinge region or both the PH domain and the hinge region.

In another embodiment with a different inositol pyrophosphate orinositol polyphosphate (or further derivatives of either), the inventionis a selective inhibitor of PDK1.

The present invention is further directed to a method of inhibiting Aktactivity which comprises administering to a mammal in need thereof apharmaceutically effective amount of the instant compound to be beprotective against TNF mediated cell apoptosis as well as sensitizecells to interferon alpha through modulations of the PI3K signalingactivity.

The compounds of the instant invention are inhibitors of the activity ofPI3-kinase/PDK1/AKT-dependent signaling pathway are thus useful in thetreatment of, cancers associated with irregularities in the activity ofAkt and/or GSK3. Such cancers include, but are not limited to ovarian,pancreatic and breast cancer. They are also useful in the treatment ofinflammatory conditions, and in the treatment of elevated Tumor NecrosisFactor in chronic inflammatory states such as like Rheumatoid Arthritis,Systemic Lupus Erythematosus, Hepatitis (A, B, C, and/or D), AnkylosingSpondylitis, Inflammatory Bowel Disease and Psoriasis, but not limitedto only these inflammatory conditions.

Chronic inflammation has been implicated in the pathogenesis of manysevere autoimmune disorders, as well as in diabetes, pulmonary diseases,and cancer. Inflammation also accompanies most solid cancers. Someresearchers investigated the role of the major proinflammatory cytokinetumor necrosis factor α(TNFα) in the malignancy of Pancreatic ductaladenocarcinoma (PDAC) cells in vitro and in vivo. In vitro, TNFαstrongly increased invasiveness of Colo357, BxPc3, and PancTuI cells andshowed only moderate antiproliferative effect. TNFα treatment of micebearing orthotopically growing PDAC tumors led to dramatically enhancedtumor growth and metastasis. Notably, they found that PDAC cellsthemselves secrete TNFα. Although inhibition of TNFα with infliximab oretanercept only marginally affected proliferation and invasiveness ofPDAC cells in vitro. In severe combined immunodeficient mice withorthotopically growing Colo357, BxPc3, or PancTuI tumors, human-specificanti-TNF antibody infliximab reduced tumor growth and metastasis byabout 30% and 50%, respectively. Infliximab and etanercept reduced thenumber of liver metastases by 69% and 42%, respectively, as well asvolumes of recurrent tumors by 73% and 51%. Thus, tumor cell-derivedTNFα appears to play a profound role in malignancy of PDAC, andinhibition of TNFα represents a promising therapeutic option for cancersthat are caused by chronic inflammation that is one cause of cancer orsolid tumors that transform to metastatic tumors. [Cancer Res 2008;68(5): 1443-50]

V. Invention Compound and Folate

In one aspect of the invention, the invention further relates toD-chiroinositol and derivatives thereof, more specificallyD-chiro-inositol, phosphates thereof, and other derivatives of each asmore fully detailed below. In addition, this aspect of the inventionalso relates to folates. The number of births presenting with spinabifida has been reduced in recent years in patients at risk of havingsuch defects by having adequate folate levels in the mother just beforeand during the first trimester of pregnancy. More specifically, if awoman takes folic acid before conception and during early pregnancy, therisk of the fetus developing a neural tube defect is reduced by about70%. Unfortunately, folate supplementation still does not prevent allsuch cases, and presumably, the impairment of the Folbp1 due to aberrantsonic hedgehog signaling likely mediates the acquisition of folateresistance of the mother. The remaining 30% risk is still substantial.In a Research Review from Neurosciences and Mental Health 2005 fromGreat Ormond Street Hospital, the use of inositol in combination withfolate therapy is mentioned as being explored. However, no particulartype of inositol is mentioned nor is any dosage amount or regimen.

Inositol prevents expression of a genetic model of neural tube defectsin mice; Nutrition Reviews, May 1997 reports that myo-inositol reducedthe incidence of neural tube defects in mouse models that are folateresistant. The curly tail model is particularly resistant to folatetherapy. (Human Reproduction, Vol. 17, No. 9, 2451-2458) and frequentlyused to test for activity in these conditions. Cogram et al, HumanReproduction, Vol. 17, No. 9, 2451-2458, states that D-chiro-inositolalone reduced frequency of spina bifida in this model to a greaterextent than myo-inositol alone. Without being bound thereto, it is theinventor's belief that in the present invention D-chiroinositol (or aphosphorylated or other derivative thereof, preferably combinations oftwo or more selected from D-chiroinositol, its phosphates, or otherderivatives thereof) stimulates these signaling mechanisms, activatingcertain isoforms of protein kinases that appear to be required forneural tube defects. Other relevant literature includes: Frederick, etal; An essential role for an inositol polyphosphate multikinase, Ipk2,in mouse embryogenesis and second messenger production. PNAS Jun. 14,2005, Vol 102, No. 24, 8454-8459; Riobo, et al. Phosphoinositide3-kinase and Akt are essential for sonic Hedgehog signaling, PNAS Mar.21, 2006, Vol. 103, No. 12, 4505-4510.

Meyers, et al; Folic Acid Supplementation and Risk for Imperforate Anusin China; American Journal of Epidemiology, Vol. 154, No. 11: 1051-1056,2001 reports on a public health campaign in China in 1993 to 1995, wherewomen were requested to take 400 mg folic acid, with or without othervitamins daily from their pre-marital examination through the end oftheir first trimester of pregnancy. The rate of imperforate anus wascalculated to be 3.1 per 10,000 births for those not taking folic acidcompare to 1.6 per 10,000 births for those taking folic acid. Theauthors conclude that folic acid may reduce imperforate anus risk.

In addition, Mo et al, Anorectal malformations Caused by Defects inSonic Hedgehog signaling, American Journal of Pathology 2001, 159,765-774 report on a mutant mouse with various defects in the SonicHedgehog signaling pathway that presents with a number of distal hindgutdefects that appear to the authors to mimic human anorectaldeformations. An excellent review of inositol and some of its phosphatesis given in Fisher, et al; Inositol and higher inositol phosphates inneural tissues: homeostasis, metabolism and functional significance;Journal of Neurochemistry, Vol 82, 736 August 2002.

In some embodiments of the present invention, folic acid of the formula

and (D)-chiro-inositol or a phosphatidyl-chiro-inositol or a complex ofthe present invention are linked via a covalent bond, preferably with anester bond formed between a carboxyl of the folic acid and hydroxyl ofthe inositol. Alternatively, with the phosphorylates or otherderivatives of the invention compounds in which the invention compoundshave a free acidic group (the —OH of a phosphate, sulfate, sulfonate, orcarbonate, the linkage can be an ester between a free hydroxy group ofthe folic acid and the acidic group of the invention compound, orfurther an acid anhydride between the folic acid acidic group and theinvention compound acidic group, although the acid anhydrides are moreprone to rapid hydrolysis. All of these compounds having an inventioncompound component and a folic acid component are further compoundswithin the invention. The inventor is using the folic acid as guidingmedia to target tumor cells with the invention compounds as described.Folic acid polysaccharides (where the polysaccharide does not contain aninositol) are analogously suitable and part of the invention formingFolic acid polysaccharide-D) chiro inositol complexes.VI. Inositols

Inositols are a group of compounds that have the following structure

where each of the R groups is either H or OH, but each carbon of thering has one H and one OH. The most common form is myo-inositol, whichis available to some degree from dietary sources. Myo-inositol requiresthat all of R1, R3, R5, R8, R9, and R12 are OH and R2, R4, R6, R7, R10,and R11 are all hydrogen. Epi-inositol and scyllo-inositol are the othertwo most abundant forms (each being substantially less than themyo-inositol in terms of abundance). D-chiro-inositol is not availablefrom dietary sources and is the isomer where R1, R3, R6, R8, R9, and R12are OH and R2, R4, R5, R7, R10, and R11 are hydrogen. In other words,D-chiro-inositol differs from myo-inositol in the inversion of R5/R6.

There are a total often isomers of inositol, and for those that havefound potential medicinal or nutritional use, many of the uses are trulylimited to particular isomers and/or phosphates thereof (where one ormore of the hydroxyl groups are phosphorylated), while for other uses(such a in connection with blood glucose regulation) more than oneinositol isomer has been found useful or is projected to be useful. Forexample, recently scyllo inositol has been found to prevent theaccumulation of amyloid β deposits and improved cognitive ability inAlzheimer's patients. (McLaurin, et al, Inositol Stereoisomers Stabilizean Oligomeric Aggregate of Alzheimer Amyloid beta Peptide and Inhibit Abeta-induced Toxicity, J. Biol. Chem., Vol. 275, Issue 24, 18495-18502,Jun. 16, 2000; and Research News from Howard Hughes medical InstituteJun. 11, 2006 A Sweet Solution to Alzheimer s Disease?) Myo-inositol wasfound not to be effective in this condition. Scyllo-inositol worked whengiven before symptoms appeared as well as after symptoms appeared inthis indication, while epi-inositol only worked at all when given beforedisease onset. Interestingly, scyllo-inositol has been reported to be an“inositol” uptake inhibitor causing similar fetal development defects innon-hyperglycemic pregnancies as seen in hyperglycemic pregnancies(Cederberg; Oxidative Stress, antioxidative defense, and Outcome inExperimental Diabetic pregnancy; Comprehensive Summaries of UppsalaDissertations from the Faculty of medicine 1008, AUU Uppsala 2001, pp.1-66). Myo-inositol has been found useful in treating panic attacks(Levine, et al, Double-blind, placebo-controlled, crossover trial forinositol treatment for panic disorder, Am J Psychiatry 1995; 152;1084-1086).

One of the more prominent uses for myo-inositol has been for blood sugarregulation. Recently, D-chiro-inositol has been proposed for insulinresistance patients (Larner, D-Chiro-Inositol—Its functional role inInsulin Action and its Deficit in Insulin Resistance, InternationalJournal of Experimental Diabetes Research 3 (2002), 47-60) on the theorythat such patients have a defect in epimerization of the myo-inositol tothe D-chiro-inositol and that the D-chiro-inositol is the active moietyin this regard. As stated above, scyllo-inositol actually resulted in anincrease in fetal defects in non-diabetic pregnancies. Phosphoinositidederangement and poor maternal metabolic turnover carries a relative riskin diabetic pregnancies for giving birth to a baby with lethalcongenital anomalies like sirenomelia (Tahna, Davari et al, 2002). Thisdetect is similar to those seen in anorectal malformation spectrum ofdefects.

OBJECTS OF THE INVENTION

It is therefore an object of the invention to provide a method ofpreventing or reducing the rate of birth defects.

It is therefore an object of the invention to use folic acid as guidingmedia for the invention compounds to target tumor cells with theinvention compounds as described within the claims where the Folic acidreceptor alpha is always expressed on a tumor. The complex will be takeninto the cell membrane by endocytosis and thereby anchor itself to aglycerophosphatide. The invention will also be made with a folicacid/inositol-PEG-liposome that will target tumor cell better than aliposome by itself.

It is therefore an object of the invention to provide a method oftreatment of women pre-pregnancy to prevent or reduce the chance offetal malformations by administering D-chiro-inositol or a phosphatederivative thereof or other derivative thereof.

It is another object of the invention to provide a method of treatmentof women during the first trimester of pregnancy to prevent or reducethe chance of fetal malformations by administering D-chiro-inositol or aphosphate derivative thereof or other derivative thereof.

It is another object of the invention to provide co-therapy for womenpre-pregnancy with both a folate source and D-chiro-inositol or aphosphate derivative thereof or other derivative thereof.

It is another object of the invention to provide a method of treatmentof women during the first trimester of pregnancy to prevent or reducethe chance of fetal malformations by co-administering D-chiro-inositolor a phosphate derivative thereof or other derivative thereof and afolate source.

It is yet another object of the invention to treat women who are takingbirth control pills but who might nonetheless become pregnant byincluding D-chiro-inositol (or a phosphate thereof or other derivativethereof) and optionally a folate source into the pills that do notcontain an estrogenic substance.

It is yet another object of the invention to treat women who are takingbirth control pills but who might nonetheless become pregnant byincluding D-chiro-inositol (or a phosphate thereof or other derivativethereof) and optionally a folate source into each of the pills in thebirth control pill packet.

It is yet another object of the invention to treat women who are takingbirth control pills and who may have excess estrogen insult withhyperactive/sensitive estrogen receptor (ER) positive breast tissue byincluding D-chiro-inositol (or a phosphate thereof or other derivativethereof) and optionally a folate source into each of the pills in thebirth control pill packet.

It is another object of the invention to prevent or reduce the rate ofbirth defects associated with in utero fetal mis-mapping.

It is yet another object of the invention to prevent or reduce the rateof birth defects associated with improper signaling in at least one ofthe sonic hedgehog, smoothened, and gli pathways.

It is still another object of the invention to prevent or reduce therate of birth defects due to a kinase disfunction in at least one of theinositol pathways.

It is another object of the invention to provide a method of preventingor reducing birth defects associated with cholesterol reductionmedications.

Yet another object of the invention is the prevention or reduction ofthe rate of birth defects associated with fetal alcohol syndrome.

It is still another object of the invention to provide a fixedcombination formulation comprising (A) at least one compound selectedfrom (a) a folate or folic acid, (b) a hormone or steroid used in birthcontrol, hormone replacement, and androgenablative therapy, (c) acholesterol lowering medication, or (d) an anticancer medication,together with (B) at least one compound selected from an inositol, aphosphorylated inositol, and derivatives thereof as defined herein.

It is still another object of the invention to treat women who are onestrogenic hormone therapy and who may have estrogen-receptor (ER)and/or, ErbB receptor overexpression phenotype mediated by the(PI3K-Akt) pathway by administering as co-therapy with said estrogenichormone therapy D-chiro-inositol (or a phosphate thereof or otherderivative thereof) thereby blocking the downstream signaling elementsresulting in cell cycle arrest in the G1 phase, thereby downregulatingthese important receptors (Breast cancer research 2004, 6: 219-224).

It is still another object of the invention to treat women who are onestrogenic hormone therapy and who may have estrogen-receptor and/or,ErbB receptor overexpression phenotype mediated by the (PI3K-Akt)pathway by administering as a single composition said estrogenic hormonetherapy drug and D-chiro-inositol (or a phosphate or other derivativethereof or other derivative thereof).

It is still another object of the invention to treat women who are onanti-androgenic hormone therapy and who may have estrogen-receptorand/or, ErbB receptor overexpression phenotype mediated by the(PI3K-Akt) pathway-receptor overexpression phenotype by administering asco-therapy with said anti-androgenic hormone therapy D-chiro-inositol(or a phosphate or other derivative thereof).

It is still another object of the invention to treat women who are onanti-androgenic hormone therapy and who may have estrogen-receptorand/or, ErbB receptor overexpression phenotype mediated by the(PI3K-Akt) pathway by administering as a single composition saidanti-androgenic hormone therapy dug and D-chiro-inositol (or a phosphateor other derivative thereof).

It is still another object of the invention to treat men who are onestrogenic hormone therapy and who may have estrogen-receptor and/or,ErbB receptor overexpression phenotype mediated by the (PI3K-Akt)pathway by administering as co-therapy with said estrogenic hormonetherapy D-chiro-inositol (or a phosphate or other derivative thereof).

It is still another object of the invention to treat men who are onestrogenic hormone therapy and who may have estrogen-receptor and/or,ErbB receptor overexpression phenotype mediated by the (PI3K-Akt)pathway by administering as a single composition said estrogenic hormonetherapy dug and D-chiro-inositol (or a phosphate or other derivativethereof).

It is still another object of the invention to treat men who are onanti-androgenic hormone therapy and who may have estrogen-receptorand/or, ErbB receptor overexpression phenotype mediated by the(PI3K-Akt) pathway by administering as co-therapy with saidanti-androgenic hormone therapy D-chiro-inositol (or a phosphate orother derivative thereof).

It is still another object of the invention to treat men who are onanti-androgenic hormone therapy and who may have estrogen-receptorand/or, ErbB receptor overexpression phenotype mediated by the(PI3K-Akt) pathway by administering as a single composition saidanti-androgenic hormone therapy drug and D-chiro-inositol (or aphosphate or other derivative thereof).

It is still a further object of the invention to reduce or prevent fetalmalformation occurrence where the fetal malformation is a neural tubedefect, a cranio-facial defect, an anorectal malformation spectrum,caudal regression syndrome, neuralectoderm derived pediatric tumors,etc.

It is still another object of the invention to provide a method ofmodulating the phosphatidylinositol/PI3K signaling pathway withcompounds and/or therapy of the present invention.

A still further object of the present invention is to provide a methodof modulating the sonic hedgehog, the receptors patched and smoothened,and GL1,2,3 transcription family pathway with compounds and/or therapyof the present invention.

Another object of the invention is to provide a method of prevention oramelioration or treatment of a phosphatidylinositol/PI3K signalingpathway signaling defect with the compounds and/or therapies of thepresent invention.

Thus, it is an object of the invention to correct the inherent mechanismof stem ell autoregulation.

Another object of the invention is to increase the efficacy of standardchemotherapeutic agents.

Yet another object of the invention is to provide a method of preventionor amelioration or treatment of a defect in the signaling pathwayassociated with sonic hedgehog, the receptors patched and/or smoothened,and/or GL1,2,3 transcription family signaling pathway with the compoundsand therapies of the present invention.

Yet another object of the invention is to provide a method or treatmentfor anti-angiogenic activity to reduce tumor incidence and/or tumorload.

Still another object of the invention is to induce antiangiogenesis inlocalized or distant metastasized tumors.

Yet another object of the invention is to decrease the risk of deep veinthrombosis (DVT's) while using chemotherapeutic agents, howeveradministered, including oral, parenteral, or transdermal, birth controlpills or hormonal products.

Yet another object of the invention is to provide a method or treatmentfor antiangoigenic activity to reduce Deep Vein Thrombosis (DVT's),pulmonary emboli (PE's) etc. utilizing the therapies and/or compounds ofthe present invention whether administered parenterally, orally,transdermally or other suitable administration mode.

Yet another object of the invention is to provide a method or treatmentfor increasing the chemotherapeutic efficacy by synergistic action ofthe current isomer (or phosphate or other derivative or of two or moreof the isomer, a phosphate thereof or other derivative thereof) withstandard chemotherapeutic agents in cancer treatments, especiallybreast, prostate, blood, colon, lung, liver, pancreatic, cervix, skin,and soft tissue cancers.

Still another object of the invention is to reduce the potentialhazardous risk of tamoxifen-associated cardiovascular disease.

Yet another object of the invention is to reduce the numbers and size oftumors locally or distant especially in breast cancers, but also cancersoriginating from blood, colon, lung, liver, cervix, prostate, skin, andsoft tissue.

Still another object of the invention is manipulating cell growth forthe regeneration of neural, hepatic, pancreatic, intestinal, spleenic,and/or cardiac tissue.

Still another object of the invention is the treatment of tissuenecrosis factor related conditions.

Yet another object of the invention is the treatment of conditionsassociated with abnormal kinase activity.

Yet another object of the invention is to protect estrogen sensitivetissue from excess estrogen insult, whether such excess insult is due toabsolute estrogen excess or relative estrogen excess with respect tonormal estrogen/androgen balance, whether endogenous or exogenouslyderived.

A further object of the invention is the prevention of or reduction inthe amount or rate of emergence of resistance of tumorigenic cells toanticancer agents.

A still further object of the invention is the administration of thecompound of the invention, with or without additional therapeutic agentsin a polymer matrix or bound to a polymer as a depot or implant.

An even further object of the invention is to inhibit cell growth in thetreatment of psoriasis, actinic keratosis, acne, dermatitis, conditionsof inappropriate or excess hair growth, and/or cosmetic purposes.

Another object of the invention is to provide methods and compositionsfor obtaining at least one of Shh loss-of-function or patched orsmoothened gain-of-function by administration of at least one inositolisomer (other than D-chiroinositol or myo-inositol), phosphorylate, orderivative thereof. Still further objects of the invention will beapparent to those of ordinary skill.

It is therefore an object of the invention to provide novel compoundsthat are inhibitors of PI3k-Akt signaling pathway.

It is also an object of the present invention to provide pharmaceuticalcompositions that comprise the novel compounds that are specificinhibitors of Akt/PKB.

It is also an object of the present invention to provide a method fortreating cancer that comprises administering such inhibitors of Akt/PKBactivity.

It is therefore an object of the invention to provide novel compoundsthat are inhibitors of overactive proinflammatory cytokines.

It is also an object of the present invention to provide a method fortreating hepatitis serous A, B, C, D that comprises administering suchinhibitors of Tumor Necrosis Factor.

It is also an object of the invention to improve the response to alphainterferon therapy by combining the invention compound with interferon.

It is another object of the invention to alleviate anemia due toenlarged spleen problems associated with chronic active hepatitis.

Yet it is another object of the invention to inhibit overexpression ofSOC3 and SHP2.

It is another object of the invention to upregilate p27kip1 to overcomeHerceptin resistance.

It is another object of the invention to upregulate P21cip for cellcycle arrest in cancer and inflammation.

Yet it is another object of the invention to down regulate or inhibitAp-1, thus inhibiting AP-1 transcription factor causes blockade ofmultiple signal transduction pathways and inhibits cancer growth.

It is another object of the invention to inhibit or downregulate ppRbthereby inhibiting tumor survival factor.

It is a still further object of the invention to provide methods,compositions, and kits that utilize activators of pyruvate kinase M2(PKM2) for the treatment, prevention, or amelioration of a disorder ordisease related to PKM2 function.

SUMMARY OF THE INVENTION

These and other objects of the invention can be achieved via theadministration, to an appropriate patient, of a compound which is amember of the family of D-chiroinositol, phosphorylates, and otherderivatives thereof. In some uses, the objects can also be achieved witha broader array of inositol based compounds, their phosphorylates, andother derivatives thereof.

The foregoing fetal malformation prevention objects and others areachieved by treating women of child bearing years with D-chiro-inositol(and/or a phosphate or other derivative thereof) and optionally a folatesource, optimally from pre-conception through at least the firsttrimester of pregnancy. Inclusion of the D-chiro-inositol along withbirth control pills has the added benefit that stores ofD-chiro-inositol (and/or phosphates and/or other derivatives thereof)and folate are high in women taking birth control pills even before theydiscontinue such treatment or become pregnant notwithstanding being onsuch therapy. A further benefit of such inclusion is thatD-chiro-inositol (or a phosphate or other derivative thereof) alsodown-regulates, modulates, or antagonizes estrogen-receptor and/or, ErbBreceptor overexpression phenotypes in breast tissue.

The breast cancer avoidance objects of the invention are achieved byadministering D-chiro-inositol (with or without folate) to patients whoare known to have or are suspect of having estrogen-receptor and/or,ErbB receptor overexpression phenotypes that is sensitive to estrogenicsubstance exposure or to anti-androgenic therapy (which may ultimatelyresult in estrogenic excess). The breast cancer avoidance (prevention)objects of the invention can be achieved in both men and women. Whilethe benefits may be greater with the folate in many of the foregoing,the benefits can also be achieved even in the absence of the folatecomponent in many of the present invention objects, and unlessspecifically excluded, or required by the context to be excluded, thefolate free methods and treatments are included within the scope of theinvention. Other objects of the invention with respect to Shhloss-of-function and/or smoothened or patched gain-of-function and thesequelae thereof are also achieved by administration of an inositolisomer other than D-chiroinositol and myo-inositol, phosphorylates,pyrophosphorylates, and other derivatives thereof and suchnon-D-chiroinositol non-myoinositol based compounds are also part of theinvention.

One aspect of the present invention makes available methods andcompositions for inhibiting certain receptors in cell pathwayactivation. In certain embodiments, the subject methods can be used tocounteract the phenotypic effects of unwanted activation of the pathway.For example, the subject method can involve contacting a cell (in vitroor in vivo) with the compositions (defined infra), such as aD-chiroinositol or a phosphate or other derivative thereof in an amountsufficient to antagonize a dependent defective pathway activation.

The invention objects involving modulating receptor-dependent pathwayactivation can be achieved by, for example, contacting a cell (in vitroor in vivo) with an agonist (defined infra) in an amount sufficient toactivate a dependent pathway activation pathway.

In general the above-mentioned inositols are selected fromD-chiroinositol, their phosphorylates, and other derivatives of eitheras described further herein. For some of the embodiments, the inositolcompound may also be a different isomer of inositol, its phosphorylatesand derivatives of either. These compounds (based on the D-chiroinositolstructure) are described more fully in co-pending U.S. patentapplication Ser. No. 11/591,398, filed Nov. 1, 2006 and U.S. Ser. No.12/001,869, filed Dec. 13, 2007. Those compounds, as well as theircorresponding analogs that differ by being based on myo-inositol orother inositol isomer rather than being based on D-chiroinositol,(whether or not further detailed herein are incorporated herein byreference) may be chosen as the inositol component in addition to any ofthe inositol component compounds specifically set forth herein. Theoptional folate component is also detailed in these two co-pendingapplications and that disclosure is also incorporated herein byreference as the optional folate component in addition to any specificdisclosure set forth herein.

The subject compounds may be formulated as a pharmaceutical preparationcomprising a pharmaceutically acceptable excipient. Antagonists of theinvention and/or preparations comprising them may be administered to apatient to treat conditions involving unwanted cell proliferation, e.g.,cancer and/or tumors (such as, without limitation, medulloblastoma,rhabdomyosarcomas, adenocarcinomas, basal cell carcinoma, etc,non-malignant hyperproliferative disorders, etc). Receptor agonists suchas those for smoothened or G-protein coupled receptors can also be usedto regulate the growth and differentiation of normal tissues. In certainembodiments, such compound or preparations are administered systemicallyand/or locally, e.g., topically, transdermally, or as an injected depotor an implant in and/or around tumor site after excision or incisionalbiopsies.

In a further aspect, the invention features a method of increasing thelevel of PKM2 activity and/or glycolysis (e.g., inhibiting theendogenous ability of a cell in the patient to down regulate PKM2) in apatient in need thereof. The method comprises the step of administeringan effective amount of an activator, preferably a selective activator,of PKM2 to the patient in need thereof, thereby increasing the level ofPKM2 activity and/or glycolysis in the patient. PKM2 is only expressedin growing cells such as cancer cells or fat cells in the patient; othertissues use other isoforms of PK. In embodiments of the invention, anactivator is used to maintain PKM2 in its active conformation orconstitutively activate pyruvate kinase activity in proliferating cellsas a means to divert glucose metabolites into catabolic rather thananabolic processes in the patient.

In another aspect, the invention features a method of regulating cellproliferation in a patient in need thereof. The method comprises thestep of administering an effective amount of an activator, preferably aselective activator, of PKM2 to the patient in need thereof, therebyregulating cell proliferation in the patient. This method can inhibitgrowth of a transformed cell, e.g., a cancer cell, or generally inhibitgrowth in a PKM2-dependent cell that undergoes aerobic glycolysis.

In another aspect, the invention features a method of treating a patientsuffering from or susceptible to a disease or disorder associated withthe function of PKM2. The method comprises the step of administering aneffective amount of an activator, preferably a selective activator, ofPKM2 to the patient in need thereof, thereby treating, preventing, orameliorating the disease or disorder in the patient. In anotherembodiment, the activator is provided in a pharmaceutical composition.

In another embodiment, the method includes identifying or selecting apatient who would benefit from activation of PKM2. The patient can beidentified on the basis of the level of PKM2 activity in a cell of thepatient (e.g., as opposed to merely being in need of treatment of thedisorder (e.g., cancer)). In another embodiment, the selected patient isa patient suffering from or susceptible to a disorder or diseaseidentified herein, e.g., a disorder characterized by unwanted cellgrowth or proliferation, e.g., cancer, obesity, diabetes,atherosclerosis, restenosis, and autoimmune diseases.

In another embodiment, the activator of PKM2 utilized in the methods andcompositions of this invention operates by or has one or more of thefollowing mechanisms or properties: the activator is an allostericactivator of PKM.2; the activator stabilizes the binding of an inositolphosphate, derivative or analog thereof in a binding pocket of PKM2; theactivator inhibits the release of sugars from a binding pocket of PKM2;the activator is an agonist, e.g., an analog, of a sugar and orphopshate e.g., an agonist which binds PKM2 with a lower, about thesame, or higher affinity than does PBP; the activator inhibits thedissolution of tetrameric PKM2; the activator promotes the assembly oftetramcric PKM2; the activator stabilises the tetrameric conformation ofPKM2; the activator inhibits the binding of a phosphotyrosine containingpolypeptide to PKM2; the activator inhibits the ability of aphosphotyrosine containing polypeptide to induce the release of a sugarphosphate from PKM2, e.g., by inducing a change in the conformation ofPKM2, e.g., in the position of Lys433, thereby hindering the release ofa native sugar of the binding pocket of PKM2; the activator binds to orchanges the position of Lys433 relative to binding pocket; the activatorselectively activates PKM2 over at least one other isoform of PK, e.g.,the activator is selective for PKM2 over one or more of PKR, PKM1, orPKL; the activator has an affinity for PKM2 which is greater than itsaffinity for at least one other isoform of PK. e.g. PKR, PKM1, or PKL;the activator has an BC.sub.50 of from about 100 micrometer to about 0.1nanomolar, e.g., about 10 micromolar to about 0.1 nanomolar, about 1micromolar to about 0.1 nanomolar, about 500 nanomolar to about 0.1nanomolar, about 250 nanomolar to about 0.1 nanomolar, about 100nanomolar to about 0.1 nanomolar, about 50nanomolar to about 0.1nanomolar, about 25 nanomolar to about 0.1 nanomolar, about 10nanomolarto about 0.1 nanomolar, about 100 nanomolar to about 1 nanomolar, about50nanomolar to about 1 nanomolar, about 25 nanomolar to about 1nanomolar, about 10 nanomolar to about 1 nanomolar; and/or the activatoris provided at a dosage of 0.1 mg to about 3000 mg per day, e.g., about1 mg to about 2400, about 15 mg to about 2400, about 15 mg to about1500, about 75 mg to about 1200, or about 75 mg to about 600 mg per day.

In another embodiment, the activator is administered at a dosage andfrequency sufficient to increase lactate production or oxidativephosphorylation.

The method may further include the step of co-administering to thepatient in need thereof an additional therapeutic agent. The term“co-administering” as used herein means that an additional therapeuticagent may be administered together with an activator of this inventionas part of a single dosage form or as separate, multiple dosage forms.Alternatively, the additional agent may be administered prior to,consecutively with, or following the administration of a PKM2 activator.In such combination therapy treatment, both the PKM2 activator and theadditional therapeutic agent(s) are administered by conventionalmethods. The administration of a composition of this invention,comprising both a PKM2 activator and an additional therapeutic agent, toa patient does not preclude the separate administration of that sametherapeutic agent, any other second therapeutic agent, or the same ordifferent PKM2 activator to the patient at another time during a courseof treatment.

When the treatment is for cancer, the additional therapeutic agent maybe a chemotherapeutic agent. When the treatment is for an autoimmunedisorder, the additional therapeutic agent may be an immune modulatoryagent. When the treatment is for obesity, the additional therapeuticagent may be a metabolic modulator like an another inositol analog. Whenthe treatment is for diabetes, the additional therapeutic agent can bean anti-diabetes drug, e.g., an oral anti-diabetes drug, e.g.,metformin, insulin, or an insulin analog or derivative. The choice of anadditional therapeutic agent will be based upon the disease or conditionthat the patient is suffering from or susceptible to, as well as thejudgment of the treating physician

In another embodiment, the patient is treated with a PKM2 activatorwithout co-administration of a hypoxic cell sensitizer, e.g.,tirapazamine.

In another embodiment, the patient is being treated for cancer ischaracterized by one or more of the following: cells in the cancer carryout aerobic glycolysis; the cancer tissue has increased glucose uptake,as compared to a control value for glucose uptake, e.g., as measured by2-deoxyglucose uptake or uptake by a labeled glucose or glucose analog;the cancer is metastatic; the cancer is PET positive; or the cancer hasincreased PKM2 expression.

In another embodiment, the activator is administered at least twice. Instill another embodiment, the activator is administered in sufficientamount and with sufficient frequency that therapeutic levels aremaintained for at least 1, 3, 5, 7, 10, 20, 30, 60, or 180 days. Inanother embodiment, the treatment is pulsatile or repeated and eachadministration provides therapeutic levels that are maintained for atleast 1, 3, 5, 7, 10, or 20 days.

In some specific embodiments, the additional therapeutic agent is aninhibitor of glutamine metabolism.

In some specific embodiments the additional therapeutic agent is aninhibitor(s) of receptor tyrosine kinase (RTK/P13K/AKT/mTOR),C-myc-hnRNPs/PKM2 network.

BRIEF DESCRIPTION OF THE DRAWING

Not Applicable

DETAILED DESCRIPTION OF THE INVENTION

I. General

A. Definitions

1. Biological and Medicinal Terms

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The phrase “aberrant modification or mutation” of a gene refers to suchgenetic lesions as, for example, deletions, substitution or addition ofnucleotides to a gene, as well as gross chromosomal rearrangements ofthe gene and/or abnormal methylation of the gene. Likewise,mis-expression of a gene refers to aberrant levels of transcription ofthe gene relative to those levels in a normal cell under similarconditions, as well as non-wild-type splicing of mRNA transcribed fromthe gene.

“Basal cell carcinomas” exist in a variety of clinical and histologicalforms such as nodular-ulcerative, superficial, pigmented, morphealike,fibroepithelioma and nevoid syndrome. Basal cell carcinomas are the mostcommon cutaneous neoplasms found in humans. The majority of new cases ofnonmelanoma skin cancers fall into this category.

“Burn wounds” refer to cases where large surface areas of skin have beenremoved or lost from an individual due to heat and/or chemical agents.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate surrounding tissues and to giverise to metastases. Exemplary carcinomas include: “basal cellcarcinoma”, which, is an epithelial tumor of the skin that, while seldommetastasizing, has potentialities for local invasion and destruction;“squamous cell carcinoma”, which refers to carcinomas arising fromsquamous epithelium and having cuboid cells; “carcinosarcoma”, whichinclude malignant tumors composed of carcinomatous and sarcomatoustissues; “adenocystic carcinoma”, carcinoma marked by cylinders or bandsof hyaline or mucinous stroma separated or surrounded by nests or cordsof small epithelial cells, occurring in the mammary and salivary glands,and mucous glands of the respiratory tract; “epidermoid carcinoma”,which refers to cancerous cells which tend to differentiate in the sameway as those of the epidermis; i.e., they tend to form prickle cells andundergo cornification; “nasopharyngeal carcinoma”, which refers to amalignant tumor arising in the epithelial lining of the space behind thenose; and “renal cell carcinoma”, which pertains to carcinoma of therenal parenchyma composed of tubular cells in varying arrangements.Other carcinomatous epithelial growths are “papillomas”, which refers tobenign tumors derived from epithelium and having a papillomavirus as acausative agent; and “epidermoidomas”, which refers to a cerebral ormeningeal tumor formed by inclusion of ectodermal elements at the timeof closure of the neural groove.

The “corium” or “dermis” refers to the layer of the skin deep to theepidermis, consisting of a dense bed of vascular connective tissue, andcontaining the nerves and terminal organs of sensation. The hair roots,and sebaceous and sweat glands are structures of the epidermis which aredeeply embedded in the dermis.

“Dental tissue” refers to tissue in the mouth which is similar toepithelial tissue, for example gum tissue. The method of the presentinvention is useful for treating periodontal disease.

“Dermal skin ulcers” refer to lesions on the skin caused by superficialloss of tissue, usually with inflammation. Dermal skin ulcers which canbe treated by the method of the present invention include decubitusulcers, diabetic ulcers, venous stasis ulcers and arterial ulcers.Decubitus wounds refer to chronic ulcers that result from pressureapplied to areas of the skin for extended periods of time. Wounds ofthis type are often called bedsores or pressure sores. Venous stasisulcers result from the stagnation of blood or other fluids fromdefective veins. Arterial ulcers refer to necrotic skin in the areaaround arteries having poor blood flow.

The term “ED₅₀” means the dose of a drug which produces 50% of itsmaximum response or effect.

An “effective amount” of, e.g., a receptor antagonist, with respect tothe subject method of treatment, refers to an amount of the antagonistin a preparation which, when applied as part of a desired dosage regimenbrings about, e.g., a change in the rate of cell proliferation and/orthe state of differentiation of a cell and/or rate of survival of a cellaccording to clinically acceptable standards for the disorder to betreated or the cosmetic purpose.

The terms “epithelia”, “epithelial” and “epithelium” refer to thecellular covering of internal and external body surfaces (cutaneous,mucous and serous), including the glands and other structures derivedtherefrom, e.g., corneal, esophegeal, epidermal, and hair follicleepithelial cells. Other exemplary epithelial tissue includes: olfactoryepithelium, which is the pseudostratified epithelium lining theolfactory region of the nasal cavity, and containing the receptors forthe sense of smell; glandular epithelium, which refers to epitheliumcomposed of secreting cells; squamous epithelium, which refers toepithelium composed of flattened plate-like cells. The term epitheliumcan also refer to transitional epithelium, like that which ischaracteristically found lining hollow organs that are subject to greatmechanical change due to contraction and distention, e.g., tissue whichrepresents a transition between stratified squamous and columnarepithelium.

The term “epithelialization” refers to healing by the growth ofepithelial tissue over a denuded surface.

The term “epidermal gland” refers to an aggregation of cells associatedwith the epidermis and specialized to secrete or excrete materials notrelated to their ordinary metabolic needs. For example, “sebaceousglands” are holocrine glands in the corium that secrete an oilysubstance and sebum. The term “sweat glands” refers to glands thatsecrete sweat, situated in the corium or subcutaneous tissue, opening bya duct on the body surface.

The term “epidermis” refers to the outermost and nonvascular layer ofthe skin, derived from the embryonic ectoderm, varying in thickness from0.07-1.4 mm. On the palmar and plantar surfaces it comprises, fromwithin outward, five layers: basal layer composed of columnar cellsarranged perpendicularly, prickle-cell or spinous layer composed offlattened polyhedral cells with short processes or spines; granularlayer composed of flattened granular cells; clear layer composed ofseveral layers of clear, transparent cells in which the nuclei areindistinct or absent; and horny layer composed of flattened, cornifiednon-nucleated cells. In the epidermis of the general body surface, theclear layer is usually absent.

“Excisional wounds” include tears, abrasions, cuts, punctures orlacerations in the epithelial layer of the skin and may extend into thedermal layer and even into subcutaneous fat and beyond. Excisionalwounds can result from surgical procedures or from accidentalpenetration of the skin.

The “growth state” of a cell refers to the rate of proliferation of thecell and/or the state of differentiation of the cell. An “altered growthstate” is a growth state characterized by an abnormal rate ofproliferation, e.g., a cell exhibiting an increased or decreased rate ofproliferation relative to a normal cell.

The term “hair” refers to a threadlike structure, especially thespecialized epidermal structure composed of keratin and developing froma papilla sunk in the corium, produced only by mammals andcharacteristic of that group of animals. Also, “hair” may refer to theaggregate of such hairs. A “hair follicle” refers to one of thetubular-invaginations of the epidermis enclosing the hairs, and fromwhich the hairs grow. “Hair follicle epithelial cells” refers toepithelial cells which surround the dermal papilla in the hair follicle,e.g., stem cells, outer root sheath cells, matrix cells, and inner rootsheath cells. Such cells may be normal non-malignant cells, ortransformed/immortalized cells.

The term “hedgehog antagonist” refers to an agent which potentiates orrecapitulates the bioactivity of patched, such as to represstranscription of target genes. Preferred hedgehog antagonists can beused to overcome a ptc loss-of-function and/or a smoothenedgain-of-function, the latter also being referred to as smoothenedantagonists. The term ‘hedgehog antagonist’ as used herein refers notonly to any agent that may act by directly inhibiting the normalfunction of the hedgehog protein, but also to any agent that inhibitsthe hedgehog signaling pathway, and thus recapitulates the function ofptc.

The term “hedgehog gain-of-function” refers to an aberrant modificationor mutation of a ptc gene, hedgehog gene, or smoothened gene, or adecrease (or loss) in the level of expression of such a gene, whichresults in a phenotype which resembles contacting a cell with a hedgehogprotein, e.g., aberrant activation of a hedgehog pathway. Thegain-of-function may include a loss of the ability of the ptc geneproduct to regulate the level of expression of Ci genes, e.g., Gli1,Gli2, and Gli3. The term ‘hedgehog gain-of-function’ is also used hereinto refer to any similar cellular phenotype (e.g. exhibiting excessproliferation) which occurs due to an alteration anywhere in thehedgehog signal transduction pathway, including, but not limited to, amodification or mutation of hedgehog itself. For example, a tumor cellwith an abnormally high proliferation rate due to activation of thehedgehog signaling pathway would have a ‘hedgehog gain-of-function’phenotype, even if hedgehog is not mutated in that cell.

As used herein, “immortalized cells” refers to cells which have beenaltered via chemical and/or recombinant means such that the cells havethe ability to grow through an indefinite number of divisions inculture.

“Internal epithelial tissue” refers to tissue inside the body which hascharacteristics similar to the epidermal layer in the skin. Examplesinclude the lining of the intestine. The method of the present inventionis useful for promoting the healing of certain internal wounds, forexample wounds resulting from surgery.

The term “keratosis” refers to proliferative skin disorder characterizedby hyperplasia of the horny layer of the epidermis. Exemplary keratoticdisorders include keratosis follicularis, keratosis palmaris etplantaris, kerawsis pharyngea, keratosis pilaris, and actinic keratosis.

The term “LD₅₀” means the dose of a drug which is lethal in 50% of testsubjects.

The term “nail” refers to the horny cutaneous plate on the dorsalsurface of the distal end of a finger or toe.

The term “patched loss-of-function” refers to an aberrant modificationor mutation of a ptc gene, or a decreased level of expression of thegene, which results in a phenotype which resembles contacting a cellwith a hedgehog protein, e.g., aberrant activation of a hedgehogpathway. The loss-of-function may include a loss of the ability of theptc gene product to regulate the level of expression of Ci genes, e.g.,Gli1, Gli2 and Gli3. The term ‘ptc loss-of-function’ is also used hereinto refer to any similar cellular phenotype (e.g., exhibiting excessproliferation) which occurs due to an alteration anywhere in thehedgehog signal transduction pathway, including, but not limited to, amodification or mutation of ptc itself. For example, a tumor cell withan abnormally high proliferation rate due to activation of the hedgehogsignaling pathway would have a ‘ptc loss-of-function’ phenotype, even ifptc is not mutated in that cell.

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human animal.

The term “prodrug” is intended to encompass compounds which, underphysiological conditions, are convened into the therapeutically activeagents of the present invention. A common method for making a prodrug isto include selected moieties which are hydrolyzed under physiologicalconditions to reveal the desired molecule. In other embodiments, theprodrug is converted by an enzymatic activity of the host animal.

As used herein, “proliferating” and “proliferation” refer to cellsundergoing mitosis.

Throughout this application, the term “proliferative skin disorder”refers to any disease/disorder of the skin marked by unwanted oraberrant proliferation of cutaneous tissue. These conditions aretypically characterized by epidermal cell proliferation or incompletecell differentiation, and include, for example, X-linked ichthyosis,psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytichyperkeratosis, and seborrheic dermatitis. For example,epidermodysplasia is a form of faulty development of the epidermis.Another example is “epidermolysis”, which refers to a loosened state ofthe epidermis with formation of blebs and bullae either spontaneously orat the site of trauma.

As used herein, the term “psoriasis” refers to a hyperproliferative skindisorder which alters the skin's regulatory mechanisms. In particular,lesions are formed which involve primary and secondary alterations inepidermal proliferation, inflammatory responses of the skin, and anexpression of regulatory molecules such as lymphokines and inflammatoryfactors. Psoriatic skin is morphologically characterized by an increasedturnover of epidermal cells, thickened epidermis, abnormalkeratinization, inflammatory cell infiltrates into the dermis layer andpolymorphonuclear leukocyte infiltration into the epidermis layerresulting in an increase in the basal cell cycle. Additionally,hyperkeratotic and parakeratotic cells are present.

The term “skin” refers to the outer protective covering of the body,consisting of the corium and the epidermis, and is understood to includesweat and sebaceous glands, as well as hair follicle structures.Throughout the present application, the adjective “cutaneous” may beused, and should be understood to refer generally to attributes of theskin, as appropriate to the context in which they are used.

The term “smoothened gain-of-function” refers to an aberrantmodification or mutation of a smo gene, or an increased level ofexpression of the gene, which results in a phenotype which resemblescontacting a cell with a hedgehog protein, e.g., aberrant activation ofa hedgehog pathway. While not wishing to be bound by any particulartheory, it is noted that ptc may not signal directly into the cell, butrather interact with smoothened, another membrane-bound protein locateddownstream of ptc in hedgehog signaling (Marigo et al., (1996) Nature384: 177-179). The gene smo is a segment-polarity gene required for thecorrect patterning of every segment in Drosophila (Alcedo et al., (1996)Cell 86: 221-232). Human homologs of smo have been identified. See, forexample, Stone et al. (1996) Nature 384:129-134, and GenBank accessionU184401. The smoothened gene encodes an integral membrane protein withcharacteristics of heterotrimeric G-protein-coupled receptors; i.e.,7-transmembrane regions. This protein shows homology to the DrosophilaFrizzled (Fz) protein, a member of the wingless pathway. It wasoriginally thought that smo encodes a receptor of the Hh signal.However, this suggestion was subsequently disproved, as evidence for ptcbeing the Hh receptor was obtained. Cells that express Smo fail to bindHh, indicating that smo does not interact directly with Hh (Nusse,(1996) Nature 384: 119-120). Rather, the binding of Sonic hedgehog (SHH)to its receptor, PTCH, is thought to prevent normal inhibition by PTCHof smoothened (SMO), a seven-span transmembrane protein. Recently, ithas been reported that activating smoothened mutations occur in sporadicbasal cell carcinoma, Xie et al. (1998) Nature 391: 90-2, and primitiveneuroectodermal tumors of the central nervous system, Reifenberger etal. (1998) Cancer Res 58: 1798-803.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

As used herein, “transformed cells” refers to cells which havespontaneously converted to a state of unrestrained growth, i.e., theyhave acquired the ability to grow through an indefinite number ofdivisions in culture. Transformed cells may be characterized by suchterms as neoplastic, anaplastic and/or hyperplastic, with respect totheir loss of growth control.

2. Chemical Terms

By “activator” is meant an agent that increases the level of activity ofPKM2 from the state of inactive monomeric or dimeric form or maintainsor increases the activity of active tetrameric form of PKM2 (e.g., inthe presence of an endogenous inhibitor). Increasing activity caninclude reducing endogenous down-regulation of PKM2 by an endogenousinhibitor (e.g., an endogenous phosphotyrosine peptide or protein). Thebinding of phosphotyrosine-containing peptide with activated PKM2results in dissociation of FBP and inactivation of PKM2. Autonomousgrowth signaling in proliferating cells or stimulation of fat cells byinsulin leads to tyrosine phosphorylation cascades. An activator canexert its effect in a number of ways including one or more of thefollowing; an activator can render PKM2 resistant to inhibition by aninhibitor, e.g., an endogenous inhibitor; an activator inhibits releaseof an activator, more specifically FBP or any inositol derivative orphosphate; an activator can bind to PKM2 and prevent an endogenousinhibitor from promoting the release of an endogenous activator, morespecifically FBP; or an activator can inhibit the dissolution or promotethe reassembly of the subunits which make up PKM2, e.g., an activatorcan inhibit oxidation of sulfhydryl moieties on such subunits, e.g.,inhibit the oxidation of cysteine residues.

An activator can cause PKM2 activity to increase to a level that isgreater than PKM2's levels (e.g., basal levels) of activity (e.g.,levels seen in the absence of an endogenous or natural activator/ligand,e.g., FBP or any inositol sugar). For example, the activator may mimicthe effect caused by an endogenous or natural ligand or activator (e.g.,FBP or any inositol sugar). The activating effect caused by the agentmay be to the same, to a greater, or to a lesser extent than theactivating effect caused by an endogenous or natural ligand oractivator, but the same type of effect can be caused. Peptides, nucleicacids, and small molecules may be activators. In preferred embodiments,the activator has a molecular weight in the range of 100 or 200 to10,000, 100 or 200 to 5,000, 100 or 200 to 2,000, or more preferably 100to 300, 200 to 500, 150 to 500, 200 to 500, 300 to 500, or 150 to 800Daltons.

Direct activators are activators which interact directly (e.g., bind) byforming a non-covalent bond such as a hydrogen, ionic, electrostatic, orhydrophobic bond, or induce a change in conformation in PKM2, includingthe tetrameric PKM2 molecule or the monomeric and dimeric molecules, oranother activator thereof. In preferred embodiments, the directactivator forms a non-covalent bond with a specific moiety on the PKM2or endogenous activor (e.g., FBP, inositol sugar/phosphate/inositolanalog thereof). Direct activators are preferred.

An expressional activator increases the expression of the PKM2 isoformat the nucleic acid level. This includes activators which induce theexpression of PKM2 at the DNA level (e.g., by acting as a co-factor toinduce transcription of PKM2) or the RNA level.

An agent can be evaluated to determine if it is an activator bymeasuring either directly or indirectly the activity of the PKM2 whensubjected to the agent. The activity of the agent can be measured, forexample, against a control substance. In some instances, directactivation of PKM2 is measured. The activity of PKM2 can be measured,for example, by monitoring the concentration of a substrate or a productdirectly or indirectly.

By “administering” is meant a method of giving a dosage of apharmaceutical composition to a patient. The compositions describedherein can be administered by a route selected from, e.g., ocular,inhalation, parenteral, dermal, transdermal, buccal, rectal, vaginal,sublingual, perilingual, nasal, topical administration, and oraladministration. Parenteral administration includes intravenous,intraperitoneal, subcutaneous, and intramuscular administration. Thepreferred method of administration can vary depending on variousfactors, e.g., the components of the composition being administered andthe severity of the condition being treated.

An “aliphatic group” includes straight chain or branched chain, orcyclic hydrocarbons (other than aromatic groups), the aliphatic grouphaving up to 30 carbon atoms (preferably up to 20 carbon atoms, morepreferably up to 10 carbon atoms, even more preferably up to 7 carbonatoms, most preferably up to 5 carbon atoms, especially methyl, ethyl,propyl, and butyl for straight chain saturated variants) and thecorresponding branched analogs and the unsaturated analogs of each and3-10 carbon atoms in the corresponding cyclic aliphatic rings(cycloaliphatic), more preferably 5, 6, or 7 ring members, Eachaliphatic group may be unsubstituted or substituted with one or moresubstituents as detailed below. Furthermore, each of the above groupscan be interrupted by one or more heteroatoms selected from nitrogen,sulfur, oxygen, and phosphorous, excepting peroxy (—O—O—). Thecycloaliphatic rings may have two, three, or four rings fused together,each ring independently having definitions in accordance with thisparagraph. Each aliphatic group and cycloaliphatic ring may beindependently unsubstituted or substituted in accordance with thedefinitions below, however, if a definition results in a continuousloop, only three loop circuits at most are permitted.

An “aromatic group” or “aryl group” as used herein includes (unlessspecifically excluded or the context requires exclusion) heteroaryls,and each ring of which has 6 to 8 ring members per aromatic ring and maybe fused to aromatic or aliphatic rings, each of which is unsubstitutedor substituted with one of more substituents as set forth more fullybelow. Heteroaryls correspond to carbocyclic aryls except that they haveone or more ring members selected from nitrogen, oxygen, or sulfur. Eacharomatic group may be independently unsubstituted or substituted inaccordance with the definitions below, however, if a definition resultsin a continuous loop, only three loop circuits at most are permitted.

Substituents for the above aliphatic and aromatic groups may include,without limitation, those from the following group (A): halogen(preferably fluorine, chlorine, bromine, or iodine, more preferablyfluorine or chlorine), hydroxyl, trihalomethyl (especiallytrifluoromethyl), cyano, carbonyl, derivatized carbonyl (such ascarboxylic acid, alkoxycarbonyl, (optionally N-substituted with alkyl oracyl)aminocarbonyl, formyl), C₂₋₇acyl, C₂₋₇acyloxy, thiocarbonyl,analogous derivitized forms thereof to the derivatized carbonyl in whichthe doubly bound oxygen is replaced by sulfur, the corresponding —C(S)SHgroup and their derivatized counterparts, phosphoryl, phosphate,phosphonate, phosphinate, amido, amidine, imine, cyano, nitro, azido,sulfhydryl, alkyl thio, sulfate, sulfonate, sulfamoyl, sulfonamide,sulfonyl, heterocyclyl (aka heterocycloaliphatic), aralkyl, aromaticgroup, or heteroaromatic group, each of which can be substituted furthernot from the above substituent list, but from one or more substituentsselected from the following group (B) consisting of amino, azido, imino,amido, phosphoryl (including phosphonate and phosphinate), sulfonyl(including sulfate, sulfonamide, sulfamoyl, and sulfonate), ether,alkylthio, carbonyl (including ketone, aldehyde, carboxylate, andester), trihalomethyl (especially trifluoromethyl), cyano, alkoxy,hydroxy, and the like, and each of these further group (B) substituentsmay be still further substituted with groups selected from group (B). Inany case where the above substituent requires a group to be specifiedthat is not so specified above or below (for example an amino, an ether,an ester, etc. where the remainder of the group cannot be determinedfrom the above or below), the preferred group is, without being limitedthereto, an alkyl of up to 7 carbon atoms or if results in a cyclicalunending definitional loop, such loop terminates after no more thanthree cycles thereof in either a hydrogen or alkyl of up to 7 carbonatoms). These substituents may also be a replacement for one or more ofthe hydrogen atoms on the inositol ring hydroxy groups or a hydroxylgroup indicated within the substituents set forth in this definition(provided that no peroxy groups result) and/or one or more of thehydrogen atoms that are on the inositol ring directly and/or or ahydroxyl group indicated within the substituents set forth in thissubstituent definition provided that if an unending loop results, suchloop terminates after no more than three cycles thereof in either ahydrogen or alkyl of up to 7 carbon atoms.

In addition to the general definition above, the term “alkylthio” refersto an alkyl group, as defined above, having a sulfur radical attachedthereto. In preferred embodiments, the “alkylthio” moiety is representedby one of —S-alkyl, —S-alkenyl, —S-alkynyl, and —S—(CH₂)_(m)-Ra, whereinm is 0-8, preferably 0-4 and R_(a) is aryl, cycloalkyl, cycloalkenyl,heterocyclyl, or polycyclyl. Representative alkylthio groups includemethylthio, ethylthio, and the like.

In addition to the general definition above, the terms “amine” and“amino” are art-recognized and refer to both unsubstituted andsubstituted amines, e.g., a moiety that can be represented by thegeneral formula:

wherein R_(b), R_(c) and R_(d) each independently represent a hydrogen,an alkyl, an alkenyl, —(CH₂)_(m)— R_(a), or carbonyl or R_(b) and R_(c)taken together with the N atom to which they are attached complete aheterocycle having from 4 to 8 atoms in the ring structure; R_(a)represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or apolycycle; and m is zero or an integer in the range of 1 to 8. Inpreferred embodiments, only one of R_(b), R_(c) and R_(d) can be acarbonyl, e.g., they and the nitrogen together do not form an imide. Ineven more preferred embodiments, each independently represent ahydrogen, an alkyl, an alkenyl, or —(CH₂)_(m)—R_(a). Thus, the term“alkylamine” as used herein means an amine group, as defined above,having a substituted or unsubstituted alkyl attached thereto, i.e., atleast one of R_(b) and R_(c) is an alkyl group.

The term “amido” is art-recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

R_(b) and R_(c) are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“hetroaryls” or “heteroaromatics.” The aromatic ring can be substitutedat one or more ring positions with such substituents as described above,for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, ether,alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl,aromatic or heteroaromatic moieties, —CF₃, —CN, or the like. The term“aryl” also includes polycyclic ring systems having two or more cyclicrings in which two or more carbons are common to two adjoining rings(the rings are “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R_(c)represents a hydrogen, an alkyl, an alkenyl, —(X_(a))_(n)—(CH)_(m)—R_(a)or a pharmaceutically acceptable salt, where X, m and R_(a) are asdefined above X_(a) is a bond or represents an oxygen or a sulfur, and nis 0 or 1, but required to be 0 when R_(c) is bound to an oxygen. When Xis oxygen and R_(c) is —(X_(a))_(n)—(CH₂)_(m)—R_(a) and X_(a) is alsooxygen, the substituent is a carbonate. Where X is oxygen and R_(c) isnot hydrogen, the formula represents an “ester”. Where X is oxygen, andR_(c), is as defined above, the moiety is referred to herein as acarboxyl group, and particularly when R_(c) is a hydrogen, the formularepresents a “carboxylic acid” or a formate respectively. In general,where the oxygen atom of the above formula is replaced by sulfur, theformula represents a “thiocarbonyl” group. On the other hand, where Xand X_(a) are each a bond, and R_(c), is not hydrogen, the above formularepresents a “ketone” group. Where X is a bond, and R_(c) is hydrogen,the above formula represents an “aldehyde” group.

By “chemotherapeutic agent” is meant a chemical that may be used todestroy a cancer cell, or to slow, arrest, or reverse the growth of acancer cell. Chemotherapeutic agents include, e.g., L-asparaginase,bleomycin, busulfan carmustine (BCNU), chlorambucil, cladribine (2-CdA),CPT11 (irinotecan), cyclophosphamide, cytarabine (Ara-C), dacarbazine,daunorubicin, dexamethasone, doxorubicin (adriamycin), etoposide,fludarabine, 5-fluorouracil (5FU), hydroxyurea, idarubicin, ifosfamide,interfron-α (native or recombinant), levamisole, lomustine (CCNU),mechlorethamine (nitrogen mustard), melphalan, mercaptopurine,methotrexate, mitomycin, mitoxantrone, paclitaxel, pentostatin,prednisone, procarbazine, tamoxifen, taxol-related compounds,6-thioguanine, topotecan, vinblastine, vincristine, cisplatinum,carboplatinum, oxaliplatinum, or pemetrexed. In another embodiment, thechemotherapeutic agent is not an anti-hypoxic agent.

By “effective amount” is meant the amount of a pharmaceuticalcomposition of the invention required to treat a patient suffering fromor susceptible to a disease, such as, e.g., cancer, diabetes, obesity,autoimmune diseases, atherosclerosis, restenosis, andproliferation-dependent diseases. The effective amount of apharmaceutical composition of the invention used for treatment variesdepending upon the manner of administration and the age, body weight,and general health of the subject. Ultimately, the attending prescriberwill decide the appropriate amount and dosage regimen. Such an amount isreferred to as the “effective amount.”

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,phosphorus, sulfur, and selenium, more preferably nitrogen, oxygen,phosphorus, and sulfur, most preferably nitrogen, oxygen, and sulfur.

The terms “heterocyclyl” or “heterocyclic group”, notwithstanding andwithout limitation to prior definitions of these terms herein, refer to3- to 10-membered ring structures, more preferably 3- to 7-memberedrings, whose ring structures include one to four heteroatoms.Heterocycles can also be polycycles. Heterocyclyl groups include, forexample, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene,xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole,isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine,isoindole, indole, indazole, purine, quinolizine, isoquinoline,quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline,cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine,pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine,furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole,piperidine, piperazine, morpholine, lactones, lactams such asazetidinones and pyrrolidinones, sultams, sultones, and the like. Theheterocyclic ring can be substituted at one or more positions with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl,carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

Also for purposes of this invention, the term “hydrocarbon” iscontemplated to include all permissible compounds having at least onehydrogen and one carbon atom. In a broad aspect, the permissiblehydrocarbons include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic organic compoundswhich can be substituted or unsubstituted. Preferably hydrocarbonscontain only hydrogen and carbon unless modified to indicate some othertype of atom is present.

By “immunomodulatory agent” is meant an agent that can elicit orsuppress an immune response. Examples of immunomodulatory agentsinclude, e.g., non-steroidal immunophilin-dependent immunosuppressants,e.g., cyclosporine (e.g., Restasis), and steroids, e.g., dexamethasone,rimexolone, fluorometholone, medrysone, and loteprednol etabonate.

By “inhibitor” is meant an agent that measurably slows, stops,decreases, or inactivates the enzymatic activity of PKM2 to a level thatis less than the PKM2's basal level of activity. Inhibitors of PKM2 maybe small molecules, peptides, or nucleic acids. Decreasing activity caninclude preventing endogenous up-modulation of PKM2 by an endogenousactivator (e.g., an inhibitor can render PKM2 resistant to activation byan activator, e.g., a naturally occurring activator and can, e.g.,promote release of an activator, e.g., FBP). In another embodiment, anactivator can promote dissolution or inhibit reassembly of the subunitswhich make up PKM2. In preferred embodiments, the activator has amolecular weight in the range of 100 or 200 to 10.000, 100 or 200 to5,000, 100 or 200 to 2,000, or more preferably 100 to 300, 200 to 500,150 to 500, 200 to 500, 300 to 500, or 150 to 800 Daltons.

Direct inhibitors are inhibitors which interact directly (e.g., bind)by, e.g., forming a non-covalent bond such as a hydrogen, ionic,electrostatic, hydrophobic or bond, or induce a change in conformationin PKM2 or a subunit or activator thereof. In preferred embodiments, thedirect inhibitor forms a non-covalent bond with a specific moiety on thePKM2 or endogenous activator (e.g., FBP). Direct inhibitors arepreferred such as any and all inositol phosphates and novel phosphatesincorporated. A direct inhibitor can be one that exerts its effect atthe protein level, or one that exerts its effect at the nucleic acidlevel. An example of the former is a compound that interacts with one orboth of PKM2 and FBP to promote release of FBP from PKM2. An example ofthe latter is a nucleic acid-based drug, e.g., an siRNA or an antisensemolecule, which targets a subunit of a PKM2.

An agent can be evaluated to determine if it is an inhibitor bymeasuring either directly or indirectly the activity of PKM2 whensubjected to the agent. The activity of the agent can be measured, forexample, against a control substance. In some instances, the activitymeasured of the agent is for inhibition of PKM2. The activity of PKM2can be measured, for example, by monitoring the concentration of asubstrate such as lactate or NADH.

As used herein, the terms “inhibit” or “prevent” include both completeand partial inhibition or prevention. An inhibitor may completely orpartially inhibit. As used herein, the term “activate” can refer todifferent levels of activation.

The term “lower” in connection with an aliphatic group means up to 7carbons, preferably up to 5 carbons, more preferably up to 4 carbons.

By “modulator” is meant an agent that modulates (e.g., activates orinhibits) the activity of pyruvate kinase (e.g., PKM2). For example, themodulator may be, e.g., a peptide that inhibits the activity of pyruvatekinase. Alternatively, a modulator may be, e.g., a nucleic acid (e.g.,siRNA) or small molecule. Modulators may be useful in the treatment of,e.g., cancer, diabetes, obesity, autoimmune diseases, neurologicaldiseases (e.g. Parkinson's disease and Alzheimer's disease),proliferation-dependent diseases, and other diseases associated with thefunction of pyruvate kinase.

By “patient” is meant any animal, e.g., mammal (e.g., a human).

By “pharmaceutical composition” is meant any composition that containsat least one therapeutically or biologically active agent and issuitable for administration to a patient. For the purposes of thisinvention, pharmaceutical compositions suitable for delivering atherapeutic can include, e.g., eye drops, tablets, gelcaps, capsules,pills, powders, granulates, suspensions, emulsions, solutions, gels,hydrogels, oral gels, pastes, ointments, creams, plasters, drenches,delivery devices, suppositories, enemas, injectables, implants, sprays,or aerosols. Any of these formulations can be prepared by well-known andaccepted methods of art. See, for example, Remington: The Science andPractice of Pharmacy (21.sup.st ed.), ed. A. R. Gennaro, LippincottWilliams & Wilkins, 2005, and Encyclopedia of Pharmaceutical Technology,ed. J. Swarbrick, Informa Healthcare, 2006, each of which is herebyincorporated by reference.

Agents useful in the pharmaceutical compositions of the invention mayinclude those described herein in any of their pharmaceuticallyacceptable forms, including isomers such as diastereomers andenantiomers, salts, solvates, prodrugs, and polymorphs, thereof as wellas racemic mixtures of the agents described herein. Which includes allinositol isomers, diastereomers, enantiomers.

A “phosphonamidite” can be represented in the general formula:

wherein R_(b) and R_(c) are as defined above, Q₂ represents O, S or N,R_(f), represents a lower alkyl or an aryl, and R_(g) represents H,lower alkyl, or aryl.

A “phosphoramidite” corresponds to the above phosphonoamidite exceptthat R_(f) is replaced by ═O:

A “phosphoryl” can in general be represented by the formula:

wherein Q₁ is O or S and R_(g) is hydrogen, a lower alkyl, or an acyl.

By “prodrug” is meant a molecule that, upon metabolism in the body of asubject, is chemically converted to another molecule serving atherapeutic or other pharmaceutical purpose (e.g., a drug moleculecontaining a carboxylic acid contains an amide or an ester bond in itsprodrug form, which is cleaved upon metabolism).

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

By “selective” is meant at least 20%, 50%, 75%, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, or 10-fold greater inhibition of a PKM2 over a secondkinase, e.g., a second pyruvate kinase, e.g., a different isoform. Thus,in some embodiments, the agent is selective for PKM2 over anotherisoform. For example, an agent is selective for PKM2 relative to PKM1.Selective regulation, e.g., inhibition or activation, or selectivemodulation, are used interchangeably with specific regulation orspecific modulation.

By “substantially identical” is meant a polypeptide or peptideexhibiting at least 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 98%, 99%, oreven 100% identity to a reference amino acid or nucleic acid sequenceover contiguous residues. Sequence identity is typically measured usinga sequence analysis program (e.g., BLAST 2; Tatusova et al., FEMSMicrobiol Lett. 174:247-250, 1999) with the default parameters specifiedtherein. Conservative substitutions typically include substitutionswithin the following groups: glycine, alanine, valine, isoleucine,leucine; aspartic acid, glutamic acid, asparagine, glutamine; sine,threonine; lysine, arginine; and phenylalanine and tyrosine.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

It will be understood that “substitution” or “substituted with” includesthe implicit provision that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

As used herein, the definition of each expression, e.g., alkyl, m, anyparticular R group, etc., when it occurs more than once in anystructure, is intended to be independent of its definition elsewhere inthe same structure.

By “therapeutic agent” is meant any agent that produces a preventative,healing, curative, stabilizing, or ameliorative effect.

By “treating” is meant administering a pharmaceutical composition forprophylactic and/or therapeutic purposes. Prophylactic treatment may beadministered, for example, to a subject who is not yet ill, but who issusceptible to, or otherwise at risk of, a particular disorder, e.g.,cancer. Therapeutic treatment may be administered, for example, to asubject already suffering from a disorder in order to improve orstabilize the subject's condition. Thus, in the claims and embodimentsdescribed herein, treating is the administration to a subject either fortherapeutic or prophylactic purposes. In some instances, as comparedwith an equivalent untreated control, treatment may ameliorate adisorder or a symptom thereof by, e.g., 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or 100% as measured by any standard technique.In some instances, treating can result in the inhibition of a disease,the healing of an existing disease, and the amelioration of a disease.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The invention described herein further features a pharmaceuticalcomposition for the treatment, prevention, or amelioration of a diseaseassociated with the function of PKM2, which comprises an activator ofPKM2 activity (e.g., a selective activator of PKM2) and apharmaceutically acceptable carrier. The activator is present in anamount that, when administered to a patient, is sufficient to treat adisease in a patient. The composition may be formulated as, e.g., apill, a powder, a granulate, a suspension, an emulsion, a solution, agel, a paste, an ointment, a cream, a foam, a lotion, a plaster, asuppository, an enema, an injectable, an implant, a spray, or anaerosol. The composition may be, e.g., formulated for targeted deliveryor for extended or delayed release. The composition may be, e.g.,formulated for oral, buccal, topical, rectal, subcutaneous, vaginal,inhalation, ophthalmic, parenteral, intravenous, or intramuscularadministration.

In some embodiments, the pharmaceutical composition further comprises anadditional therapeutic agent useful in the treatment of a patientsuffering from or susceptible to a disease or condition selected fromcancer, atherosclerosis, restenosis, an autoimmune disorder, aproliferative disorder, or obesity. In a more specific embodiment, theadditional therapeutic agent is selected from a chemotherapeutic agent,an immune modulatory agent, a metabolic modulator, an anti-diabetesdrug, insulin, or an insulin analog or derivative.

The invention described herein features a kit that includes apharmaceutical composition containing a PKM2 activator and instructionsfor administering the composition to a patient having a diseaseassociated with the function of PKM2. The kit may further include anadditional therapeutic agent. The additional therapeutic agent will beappropriate for the disease or condition to be treated by the kit, andmay be selected, e.g., from any of the additional therapeutic agents setforth above for combination therapies.

In another aspect, the invention features a method for evaluating acandidate compound for the ability to activate PKM2. The method includesproviding a PKM2 polypeptide which includes at least the FBP bindingregion of PKM2; contacting the PKM2 polypeptide and the candidatecompound assessing the ability of the candidate compound to activatePKM2; and optionally, determining if the candidate compound binds toPKM2, thereby evaluating a candidate compound for the ability toactivate PKM2.

In another embodiment the ability of the compound to activate PKM1, PKR,or PKL is determined and compared with the ability of the candidatecompound to activate PKM2.

In yet another embodiment, the PKM2 polypeptide is a polypeptide presentin human PKM2. It can include one or more of the following human PKM2amino acids: Thr432, Lys433, Ser434, Ser437, Trp482, Arg489, Gly514,Gly518, Ser519, Gly520, and Phe521 (e.g., residues identified bycrystallographic studies that form salt bridges and hydrogen bonds withFBP), and/or K433, D488, R489, R455, T454, T434, and N456 (e.g.,residues that potentially define the phosphotyrosine binding pocket) orother Pleckstrin Homology-pH domain pockets. In another embodiment, thepolypeptide includes all or a portion of the PKM2 sequence fromT432-G514. In other preferred embodiments, the PKM2 polypeptide includessufficient sequence to allow FBP binding or FBP binding, orinositol/phosphatebinding and phosphotyrosine-modulated release of asugar anlog/phosphate or FBP. In another embodiment, the PKM2polypeptide includes the entire PKM2 sequence. Preferably, the PKM2polypeptide is present as a tetramer.

In another embodiment, contacting the PKM2 polypeptide and the candidatecompound can include: forming a reaction mixture (e.g., a cell-freemixture) containing the PKM2 polypeptide, which can, e.g., be purifiedor partially purified, and the candidate compound; contacting a cellthat expresses the PKM2 polypeptide, e.g., a cancer cell, with thecandidate compound; or administering the candidate compound to an animalthat expresses the PKM2 polypeptide.

In a particular and specific embodiment, a reaction mixture is formedand includes FBP or inositol/phosphates or analogs. Such embodiments areuseful in evaluating compounds that activate PKM2 by inhibiting therelease of FBP or sugars from PKM2.

In another specific embodiment, the reaction mixture excludes FBP. Suchembodiments are useful in evaluating compounds which mimic or areagonists of FBP.

In another embodiment, the reaction mixture includes one or more ofsubstrate, cofactor, buffer, and assay or readout reagents.

In another embodiment, a reaction mixture is formed and includes FBP andphosphotyrosine peptide, or inositol phosphates/analogs andphosphotyorosine peptide or mixtures of all. Such embodiments are usefulin evaluating the candidate compound's ability to activate PKM2 byinhibiting the release of FBP from PKM2 in the presence ofphosphotyrosine peptide.

In another embodiment, assessing includes the step of evaluating thelevel of a substrate consumed or a product produced by a reactioncatalyzed by PKM2 directly or indirectly. This can include measuring theproducts of a PKM2 reaction, e.g., ATP or pyruvate, or in a coupledreaction with the presence of lactate dehydrogenase measuring theconsumption of NADH and/or the production of lactate. In specificembodiments, the readout of the assessment is made spectroscopically,e.g., colorimetrically or fluorometrically. In another embodiment, thelevel or rate of consumption, production is compared with a positivecontrol. If the level or rate is equal to or greater than the control,the candidate compound is selected.

In another embodiment, assessing includes using labeled reagents, e.g.,a radioisotope-labeled glucose, and scintillation counting to follow thefate of that reagent. Assessing can include measuring PKM2 activitydirectly by measuring the consumption of ADP or phosphoenolpyruvate, orby measuring the production of ATP or pyruvate. These measurements maybe made spectroscopically or by any other method. Production of ATP canalso be measured using luminescence by coupling the PKM2 reaction to theluciferase reaction. A change in cellular oxygen consumption can also bemeasured.

In another embodiment, assessing includes using a coupled enzymereaction in the presence of a second enzyme that utilizes the product ofpyruvate kinase reaction (pyruvate and ATP). In another embodiment, thesecond enzyme is lactate dehydrogenase which converts pyruvate tolactate in the presence of NADH. In another embodiment, the productionof ATP can be measured by a bioluminescence ATP assay.

In another embodiment, the observed ability of the candidate to activatePKM2 is compared with a control or preselected value, and if theobserved ability meets a preselected relationship with the control orpreselected value, e.g., it meets or exceeds, the candidate compound isselected for further analysis.

Further analysis can include confirming that the candidate compoundactivates PKM2. In one embodiment, the method further includesperforming a second evaluation for the ability to activate PKM2 by thesame method. In another embodiment, the method further includesperforming a second evaluation for the ability to activate PKM2 by adifferent method. In certain embodiments, the first method is acell-free system and the second is a cell-based assay. In alternateembodiments, the first method is a cell-free or cell-based method andthe second method is an animal-based method.

In a specific embodiment, the confirmatory assay includes the step ofperforming a second evaluation for the ability of the candidate compoundto activate PKM2 by contacting the candidate compound with a cell andmeasuring the consumption of oxygen or production of lactate by thecell. In other specific embodiments, a decrease in any of cellularphosphoenolpyruvate, glycerol-phosphate, ribose or deoxyribose, lipidsynthesis, or glucose conversion to lipid or nucleic acids or aminoacids or protein by the cell can be used to confirm the ability of thecandidate compound to activate PKM2. The evaluation could includemeasuring an increase in pyruvate but this is hard to measure in acell-based assay. The measurement could also determine alteration inmitochondrial membrane potential, e.g., as measured by fluorescentpotentiometric dyes.

In certain embodiments, the confirmatory assay employs an animal-basedassay, e.g. one which uses a mouse or rat, and which allows assessmentof the ability to activate PKM2 in the animal. In some embodiments, thecandidate compound is contacted with a test animal and the conversion of¹³C-labeled glucose to pyruvate or lactate or ribose or othermetabolites is followed by MRI in vivo or by mass spectrometry ofmetabolites from extracted tissues. In certain embodiments, the animalmodel is evaluated by a method which monitors glucose uptake, e.g., aPET or MRI scan.

In specific embodiments, the candidate compound has one or moreproperties described herein, e.g., one or more of the followingproperties: the candidate compound is an allosteric activator, thecandidate compound inhibits the release of FBP; the candidate compoundis an agonist of FBP, e.g., an agonist which binds with a lower, aboutthe same, or higher affinity than does FBP; the candidate compound whichcan be ANY and ALL inositol derivatives and their phosphates, andinositol analogs thereof incorporated by reference herein, inhibits thedissolution of tetrameric PKM2; the candidate compound promotes theassembly of tetrameric PKM2; the candidate compound selectivelyactivates PKM2 over at least one other isoform of PK, e.g., thecandidate compound is selective for PKM2 over PKR, PKM1, or PKL; or thecandidate compound has an affinity for PKM2 which is greater than itsaffinity for at least one other isoform of PK, e.g., PKR, PKM1, or PKL.In another embodiment, the method of evaluating the ability of acandidate compound to activate PKM2 further includes evaluating thecandidate to determine if it has one of the properties described herein.

In another embodiment, more than one candidate compound is evaluatedsimultaneously.

In another embodiment, the method of evaluating the ability of acandidate compound to activate PKM2 includes the step memorializing theoutcome of an evaluation or assay described herein.

In another aspect, the invention features a method of evaluating acandidate structure for its ability to interact with PKM2. The methodcan be used to evaluate a candidate structure for use or furtherinvestigation for use as an inhibitor or activator of PKM2. The methodincludes: providing a three dimensional representation of a PKM2structure, which includes a portion of PKM2 including the FBP bindingpocket and preferably Lys433; providing a three dimensionalrepresentation of a candidate structure; and evaluating a relationship,e.g., fit, distance, or spatial overlap, between the PKM2 and candidatestructures, or between an atom, amino acid, or moiety on the PKM2structure and an atom or moiety on the candidate structure, therebyevaluating the candidate structure for its ability to interact withPKM2.

The crystal structure of PKM2 complexed with FBP has been reported (see,e.g., Dombrauckas et al., Biochemistry 44:9417-29, 2005, incorporated byreference herein.

Art-known methods can be used to generate three-dimensionalrepresentations of molecules for which the structure is provided orwhich have been purified or crystallized. Art known methods can be usedto produce computer-generated simulations which allow structuralcomparisons, such as the ability of a candidate structure to “dock” withPKM2.

In another embodiment, a three-dimensional structure can be generated bya modeling program which predicts the three-dimensional structure, forexample, from the primary sequence of a protein or peptide. In anotherembodiment, a three-dimensional structural representation can begenerated from a crystal structure.

In another embodiment, the conformation of activator molecules bound toPKM2 can be obtained from NMR measurements of the co-complex of a smallmolecule activator and PKM2. In some embodiments, the PKM2 structure isprovided for a PKM2 bound to FBP. In other embodiments, the PKM2structure provided is that of PKM2 without bound FBP. In someembodiments, the PKM2 structure provided is bound to aphosphotyrosine-containing polypeptide.

In some embodiments, evaluating includes determining the distancebetween an atom or moiety of the candidate structure and an atom ormoiety of a residue of PKM2, e.g., of a residue in or near, e.g., within5 angstroms of, the FBP binding pocket, for example, determining if anatom or moiety of the candidate structure and an atom or moiety on theFBP binding pocket make contact or come within a pre-selected distanceof one another.

In some embodiments, evaluating includes evaluating the relationship ofan atom or moiety of the candidate structure with an atom or moiety ofthe binding pocket, an atom or moiety of PKM2 within 5 angstroms of thebinding pocket, an atom or moiety of FBP or inositol phosphate residingin the binding pocket, or with an atom or moiety of Lys433.

In some embodiments, evaluating includes determining whether thecandidate structure displaces a ligand in the FBP binding pocket or PHdomain pocket or would result in steric hindrance with a bound ligand,e.g., FBP, in the FBP pocket or PH domain. In other embodiments,evaluating includes determining whether the candidate structure wouldinterfere with occupancy of a ligand in the FBP binding pocket. In otherembodiments, evaluating includes determining whether the candidatestructure displaces a ligand in a binding pocket or would interfere withrelease of a ligand in the FBP binding pocket or another binding pocket.

In some embodiments, evaluating includes determining whether a shift ofamino acid K433 occurs when fitting or docking the PKM2 and candidatestructures. In some embodiments, the evaluating includes determiningwhether one or more interactions occur between the two structures.Exemplary interactions include hydrogen bonding, formation of a saltbridge, hydrophobic interactions, and hydrogen interactions.

In some embodiments, the method further includes making a record of theevaluation, for example, in a tangible medium such as computer memory oron paper. In another embodiment, the record includes an identifier forthe candidate structure and a value for a parameter related to therelationship evaluated.

In another embodiment, the method further includes providing a secondcandidate structure and repeating one or more of the above recited stepson the second candidate structure. In another embodiment, theevaluations for the first and second candidate structures are comparedand one is selected for further analysis.

In another embodiment, the method further includes providinginstructions to synthesize, purchase, or otherwise obtain a candidatestructure evaluated by the method. The identity of a candidate to besynthesized, purchased, or otherwise obtained can be memorialized bycreating a record of the identity of the candidate, for example, in atangible medium such as computer memory or on paper.

In another embodiment, the candidate is tested for its ability tointeract with PKM2.

In another aspect, the invention features a pharmaceutical compositionof any of the activators described herein.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

3. Compounds

In the following discussion, for the invention compounds, reference willbe made to D-chiroinositol, its phosphorylates, and derivatives ofeither, but it should be understood that the present invention, unlessspecifically indicated otherwise, or the context requires or theavoidance of prior art requires, includes the corresponding compoundsbased on any other inositol isomer. Where exclusion is not explicit inthis specification, but is required due to prior art, the inventioncompounds are to be deemed to exclude such prior art compounds inparticular, and preferably exclude compounds having the same substituentpattern exactly as the prior art compounds but based on an inositolisomer other than D-chiroinositol or myoinositol, or preferably havingthe same substituent pattern as the prior art compound but based onatrinositol isomer other than D-chiroinositol, or preferably having thesame substituents but in the same pattern or a different pattern fromthat in the prior art molecule based on the same inositol isomer as theprior art compound, or preferably having the same substituents but inthe same pattern or a different pattern from that in the prior artmolecule based on an inositol isomer other than D-chiroinositol ormyo-inositol, or preferably having the same substituents but in the samepattern or a different pattern from that in the prior art molecule basedon an inositol isomer other than D-chiroinositol. These exclusions areon a utility-by-utility and composition-by-composition basis so thatexclusion of a compound for one utility does not exclude the compoundfrom being within the invention for another utility or exclusion of acompound in one formulation does not exclude the compound for beingwithin the invention for another composition. Every recitation ofcompounds in this specification as part of the invention is deemed to bethe specific recitation as well as the more general recitations toinclude the corresponding compounds based on inositol isomers other thanthe one specifically mentioned subject to the exclusions set forth inthis paragraph, and on the location of prior art that requires exclusionof one or more compounds on a particular use or composition, applicationof the various exclusions above to the particular recitation in theparticular context is deemed to have been specifically recited herein.

4. Uses

In one aspect, the present invention relates to the discovery thatsignal transduction pathways regulated by either phosphatidylinositoland/or by Shh and its constituents (patched (ptc), gli and/orsmoothened) can be inhibited, at least in part, by D-chiroinositoland/or derivatives thereof (as set forth more fully below). While notwishing to bound by any particular theory, the activation of thereceptor proteins is believed to be the mechanism by which these agentsact. For example, the ability of these agents to inhibit proliferationor patched loss-of function (ptclof) cells may, be due to the ability ofsuch molecules to interact with hedgehog, patched, or smoothened, or atleast to interfere with the ability of those receptor proteins toactivate a hedgehog, ptc, and/or smoothened-mediated signal transductionpathway. Again, without being bound thereto, it is the inventor's beliefthat D-chiroinositol (or a phosphorylated or other derivative thereof,preferably combinations of two or more selected from D-chiroinositol,its phosphates, or other derivatives thereof) stimulates these signalingmechanisms, activating certain isoforms of protein kinases which kinasesappear to be involved in neural tube defects prevention.

It is, therefore, specifically contemplated that these small molecules(D-chiroinositol and its derivatives) which interfere with aspects ofsignal transduction activity will likewise be capable of inhibitingproliferation (or other biological consequences) in cells. In preferredembodiments, the subject inhibitors are organic molecules having amolecular weight less than 2500 amu, more preferably less than 1500 amu,and even more preferably less than 750 amu, and are capable ofmodulating (inhibiting or activating) at least some of the biologicalactivities of hedgehog proteins, protein kinases and preferablyspecifically in target cells.

Thus, the methods of the present invention include the use of D-chiroinositol and/or derivatives thereof (optionally with folate sources)which agonize (mimic) the inhibition of certain receptor complexes ofhedgehog signaling, such as by inhibiting activation of downstreamcomponents of the signal pathway, in the regulation of repair and/orfunctional performance of a wide range of cells, tissues and organs. Forinstance, the subject method has therapeutic and cosmetic applicationsranging from regulation of neutral tissues, bone and cartilage formationand repair, regulation of spermatogenesis, ovulation, regulation ofsmooth muscle, regulation of lung, liver and other organs arising fromthe primitive gut, regulation of hematopoietic function, regulation ofskin and hair growth, etc. Moreover, the subject methods can beperformed on cells which are provided in culture (in vitro), or on cellsin a whole animal (in vivo). See, for example, PCT publications WO95/18856 and WO 96/17924 (the specifications of which are expresslyincorporated by reference herein).

In a preferred embodiment, the subject method can be to treat epithelialcells. For instance, the subject method can be used in treating orpreventing basal cell carcinoma or other hedgehog pathway-relateddisorders.

In another preferred embodiment, the subject method can be used as partof a treatment regimen for malignant medulloblastoma and other primaryCNS malignant neuroectodermal tumors. Additional exemplary cancers inwhich the present invention is of use includes those in the followingtable

ORGAN OR TISSUE SPECIES CELL LINE 1. Blood Human Erythrolekemia K562cell line K562 + human bone marrow 2. Colon Human AdenocarcinomaHT-29cell line 3. Lung Rat Tracheal epithelium + B{a}P 4. Liver HumanHepG2 cells 5. Mammary Human Adenocarcinoma MCF-7, MDA-MB 231 cells 6.Cervix (uterine) Human HeLa cells 7. Skin Mouse JB6 cells Mouse HEL-30cells 8. Soft tissue Mouse 3T3 fibroblast Human Rabdomyosarcoma, RDcells (Shamsuddin Abul& Vucenik, Ivana, Current Cancer Therapy Reviews,2005, 1, 259-269).

In another aspect, the present invention provides pharmaceuticalpreparations comprising, as an active ingredient which isD-chiroinositol or a derivative thereof with or without a folate source,a receptor antagonist/agonist, or protein modulator,(antagonist/agonist), a kinase antagonist or agonist formulated in anamount sufficient to inhibit, in vivo, proliferation or other biologicalconsequences of aberrant PI3K/Shh cellular signaling, especially ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function.

The subject treatments can be effective for both human and animalsubjects. Animal subjects to which the invention is applicable extend toboth domestic animals and livestock, raised either as pets or forcommercial purposes. Non-limiting examples are dogs, cats, cattle,horses, sheep, hogs, and goats.

The present invention is further a method of treatment so as to avoid orreduce the incidents of fetal malformations and the avoidance orreduction of activation of breast cancer (or breast cancer precursorcondition) in either men or women. The treatment and compositions can beadministered to men or women are on estrogenic hormonal therapy oranti-androgenic hormonal therapy (which results in anestrogenic/androgenic balance of surplus of estrogenic-receptor effects)or to those with known or suspect highly estrogen sensitive epithelialand/or mammary breast tissue.

One of the significant aspects of the invention is the prevention of orreduction of the risk of birth defects associated with fetal alcoholsyndrome, low cholesterol levels during pregnancy, and/or protein kinaseoveractivity. The method involves administering to a woman during herpregnancy a compound selected from the group consisting of an inositol,a phosphorylate thereof, a derivative of either (as described more fullybelow), or mixtures thereof. The administration during the firsttrimester is of particular value, especially because the fetus is mostsensitive to aberrations at this time. As such, it is preferred that thewomen be treated prophylactically in order to assure treatment when shemay not realize she is pregnant and therefore may be likely to beconsuming alcohol or be treated with cholesterol lowering medicationswithout thinking of the issues of impact on a fetus.

Without being bound to theory, it is the inventor's belief that exposureto alcohol during the critical period during first trimester embryonicdevelopment may inhibit efficient transcription of genes, especiallythose related to expression of proteins involved in proper mappingduring fetal development. This in turn leads to impaired signaltransduction during fetal mapping. Also, mutations in genes that encodespecific kinases that produce phosphate-responsive promoters for propergene expression may also be impaired during alcohol exposure. It is alsothe inventor's belief that alcohol induces hyperactive active states ofgene encoding phosphate kinases which may contribute to defective orloss of shh signaling. Therefore, supplementing alcohol exposed fetusesby administering at least one of an inositol (especially myo-inositoland/or D-chiroinositol, particularly D-chiroinositol) and/or aphosphorylate thereof (especially a polyphosphorylate thereof as furtherdefined herein) and/or a derivative of the foregoing (as further definedherein) optionally in the presence of a folate source (preferably folicacid) (to inhibit hyperactive states of protein kinases) should restoreproper signaling and prevent major birth defects related to fetalalcohol exposure. In addition, supplementing the embryos with thesecompounds will also rescue the loss of proper shh gene expression and/orinhibit hyperactive states of certain protein kinases that impairtranscription factors. This leads to the use of these compounds for theprevention of these common congenital defects related to fetal alcoholsyndrome.

Again, without being bound to theory, the present inventor believes thatthe low cholesterol levels also result in a negative impact on signalingand is the basis for the thought that statins might be teratogenic andtherefore contraindicated during pregnancy. The inventor believes thatsupplementation with the above inositols (the free inositols,phosphorylates thereof and/or derivatives of either or mixtures thereof)optionally in the presence of a folate source (preferably folic acid)can restore (at least in part) proper mapping function in thoseindividuals who have low cholesterol levels, either naturally or havelow cholesterol levels due to use of cholesterol lowering medicationssuch as statins and/or fibrates.

In the foregoing situations, the use of the above compounds (inositols,phosphorylates thereof, and/or other derivatives of either as definedherein) are useful for the prevention or reduction in incidence of fetalalcohol syndrome defects (FASD) as well as safeners for the use ofalcohol or cholesterol lowering medications in women who are pregnant aswell as those who may be pregnant and not yet be aware of the pregnancy.

Administration of the above compounds is clearly indicated when thepatient is a women of child bearing age and is known to be pregnant. Itis especially of use when the patient is known to be a frequent user ofalcohol and is not a candidate to adhere to an alcohol free pregnancy.However, since it is unclear as to whether even infrequent alcohol usecan give rise to FASD, it is preferable to generally use the abovecompounds in a prophylactic general manner with all women who arepregnant so as to minimize the rate of FASD overall. In addition, sincea particularly sensitive time period for the fetus is the early stagesof pregnancy (first trimester, especially the first month) and this isthe precise period when a woman is least likely to know she is pregnant,it is the most likely time when fetal injury can occur due to alcoholand/or depressed cholesterol. Thus, the inventor believes that the abovecompounds should be used prophylacticly in all women of child bearingage who utilize alcohol on a regular basis and in all women of childbearing age who are either on cholesterol lowering medications or whonaturally have abnormally low cholesterol levels.

The subject methods and compounds may be used to regulate proliferationand/or differentiation of cells in vitro and/or in vivo, e.g., in theformation of tissue from stem cells, or to prevent the growth ofhyperproliferative cells to illustrate but a few uses. For example,according to the present invention, large numbers of non-tumorigenicneural progenitor cells can be perpetuated in vivo and theirdifferentiation and proliferative rates can be amplified preferably inthe presence of growth factors by contacting the cells with the subjectcompound.

II. Particulars

A. Compounds

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including, cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, especially theracemic mixtures (racemates) thereof, and other mixtures thereof, asfalling within the scope of the invention, except that chiro inositoland the phosphates and derivatives thereof used in the present inventionhave the inositol ring (i.e. a six carbon 6 membered ring, with eachcarbon having one H and one OH in the underivitized form) and especiallyin the form of D-chiroinositol. In some embodiments this limitation tothe D-chiro form of inositol (its phosphorylates and derivatives ofeither) is to the exclusion of the other isomeric forms of inositolregardless of the actual naming convention of the complete molecule. Inother embodiments of the invention, the invention requires an inositol,a phosphorylate thereof or a derivative of either without being limitedto the D-chiroinositol structural form. In some contexts, this will bespecifically pointed out and in others, as indicated in the definitionssection of this specification, the exclusion of compounds will be on thebasis that such compounds are part of the prior art in a limited numberof contexts. Additional asymmetric carbon atoms may be present in asubstituent such as an alkyl group. All such isomers, as well asmixtures thereof, are intended to be included in this invention.

For convenience, the structure of D-chiroinositol is as follows and thenaming convention used herein follows that shown, regardless of whetherthe IUPAC naming convention differs for this particular compound or itsderivatives:

In the substituted compounds of the invention, to the extent that thecompounds retain either hydroxyl groups or have hydroxyl groups in whichthe hydrogen of the hydroxyl has been replaced, the retention of thespecial arrangement of the shown hydroxyl groups by the retainedhydroxyls or the inositol ring carbon-Oxygen bond of the derivatizedcompound or the inositol ring carbon-carbon or other heteroatom bondreplacing one or more of the D-chiroinositol hydroxyl groups defines aD-chiroinositol compound. Replacement of the inositol ring hydrogenswill not affect the consideration of the compound as a D-chiroinositolderivative for this application. Thus, in each of the exemplarycompounds below where the hydroxy group or derivatized hydroxy grouparrangement mimics D-chiro inositol, it is a D-chiroinositol

compound of the invention. Where a hydroxyl is replaced by a hydrogenand the other hydrogen is replaced by another group (see the finalstructure above of this paragraph), then it is not a D-chiroinositolderivative for the present invention, although it is a derivitizedinositol and may be within the scope of compounds for other aspects ofthe invention. However, where both the hydrogen and the hydroxyl of aparticular D-chiroinositol ring carbon atom are replaced by substituents(see the structures below), both are to be considered D-chiroinositolderivatives for purposes of the invention regardless of the actualnaming convention name, except in the case where one or both are foundin the prior art, in which case the prior art compound is not to beconsidered a D-chiroinositol derivative for the present invention and issubject to the exclusionary aspects indicated above.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts may be formed with an appropriateoptically active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., the ability to inhibit hedgehogsignaling), wherein one or more simple variations of substituents aremade which do not adversely affect the efficacy of the compound. Ingeneral, compounds which themselves are not inositols, inositolphosphorylates or other inositol derivatives are not consideredequivalents for the present invention. The compounds of the presentinvention may be prepared by the methods illustrated in the generalreaction schemes as, for example, described below, or by modificationsthereof, using readily available starting materials, reagents andconventional synthesis procedures. In these reactions, it is alsopossible to make use of variants which are in themselves known, but arenot mentioned here.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CRC Handbook ofChemistry and Physics, 82th Ed., 2001-2002, inside cover.

B. Exemplary Compounds of the Invention.

As described in further detail below, it is contemplated that some ofthe subject methods can be carried out using any of D-chiroinositol,D-chiroinositol phosphates, or a variety of different D-chiroinositolderivatives or mixtures thereof which can be readily identified, e.g.,by such drug screening assays as described herein. Other aspects of theinvention can be carried out by these same D-chiroinositols as well asthe corresponding inositols which are based on inositol isomers otherthan the D-chiro form. These other inositol forms when underivitized,differ from D-chiroinositol only in the particular orientation of thehydroxy groups.

D-chiro-inositol is a compound of the structure I

D-chiro inositol is not present in dietary sources and is derived fromsoil. Any such compound available to the body must be made by conversionof other sources, either systemically or artificially. The most commonsource of inositols is myo-inositol, which does occur in dietarysources. Myo-inositol differs from D-chiro-inositol by inversion of theOH and H at the position indicated by the arrow in Figure I above.Methods of making D-chiro-inositol are detailed in a number of patents,among them, U.S. Pat. Nos. 5,091,596; 5,406,005; 5,463,142; 5,714,643,5,932,774; and 6,660,891, all of which are incorporated herein byreference. Phosphates thereof for purposes of the present inventioninclude those having one or more of the hydroxyl groups in formula Iabove phosphorylated. These include mono-, di-, tri-, tetra-, penta-,hexa-monophosphates, and heptaphosphates. For convenience, thephosphates of D-chiroinositol will be referred to herein by the termD-chiroIP_(x), where x refers to the number of phosphorylated hydroxylgroups that are present. Where there is one or more numbers present asin 1,2-D-chiroIP₂, the designation indicates the position of thephosphate(s) based on the position numbering in Figure I above. Adesignation such as 1,2-D-IP₃ indicates that positions 1 and 2 arephosphorylated and that another position is phosphorylated, but that itcan be at any other position. The absence of any numerical designationbefore the “IP” indicates that the phosphate groups are not restrictedto any particular position(s). The use of the term “IP” without thedesignation “D-chiro” shall mean that inositol phosphates more generallyand include phosphorylated forms of any isomeric form of inositol.Specific mention of particular isomeric forms of inositol, such as myo-,or scyllo-, epi-, etc with the “IP_(x)” designation shall refer only tothat particular inositol isomer phosphorylated in accordance with thenumeric prefix and “x” designation in the foregoing convention. Thus,the present invention relates to compositions and methods of use ofD-chiroinositol, its monophosphates (D-chiroIP₁), diphosphates(D-chiroIP₂), triphosphates (D-chiroIP₃), tetraphosphates (D-chiroIP₄),pentaphosphates (D-chiroIP₅), and hexaphosphate (D-chiroIP₆). A compoundindicated as D-chiroIP₇ indicates a D-chiroinositol having 1pyrophphosphate and 5 monophosphate groups or 2 pyrophosphate groups and3-monophosphate groups, or 3-pyrophosphate groups and one monophosphategroups. Higher subscripts for the IP indicate similar multiplepyrophosphate and/or monophosphate groups. While polyphosphates of 3 ormore linked phosphates (such as —O—P(O)(OH)—O—P(O)(OH)—O—P(O)(OH)—O—)are possible, they are more prone to rapid hydrolysis and therefore lessdesirable. D-chiroinositol has 6 distinct monophosphates, 15 distinctdi(mono)phosphates, 20 distinct tri(mono)phosphates, 15 distincttetra(mono)phosphates, 6 distinct penta(mono)phosphates, and 1hexaphosphate, each of which are intended to be included within thescope of the present invention (unless otherwise noted or the contextcompels otherwise). These are 1-D-chiroIP₁, 2-D-chiroIP₁, 3-D-chiroIP₁,4-D-chiroIP₁, 5-D-chiroIP₁, 6-D-chiroIP₁, 1,2-D-chiroIP₂,1,3-D-chiroIP₂, 1,4-D-chiroIP₂, 1,5-D-chiroIP₂, 1,6-D-chiroIP₂,2,3-D-chiroIP₂, 2,4-D-chiroIP₂, 2,5-D-chiroIP₂, 2,6-D-chiroIP₂,3,4-D-chiroIP₂, 3,5-D-chiroIP₂, 3,6-D-chiroIP₂, 4,5-D-chiroIP₂,4,6-D-chiroIP₂, 5,6-D-chiroIP₂, 1,2,3-D-chiroIP₃, 1,2,4-D-chiroIP₃,1,2,5-D-chiroIP₃, 1,2,6-D-chiroIP₂, 1,3,4-D-chiroIP₃, 1,3,5-D-chiroIP₃,1,3,6-D-chiroIP₃, 1,4,5-D-chiroIP₃, 1,4,6-D-chiroIP₃, 1,5,6-D-chiroIP₃,2,3,5-D-chiroIP₃, 2,3,6-D-chiroIP₃, 2,4,5-D-chiroIP₃, 2,4,6-D-chiroIP₃,2,5,6-D-chiroIP₃, 3,4,5-chiroIP₃, 3,4,6-D-chiroIP₃, 3,5,6-D-chiroIP₃,4,5,6-D-chiroIP₃, 1,2,3,4-D-chiroIP₄, 1,2,3,5-D-chiroIP₄,1,2,3,6-D-chiroIP₄,1,2,4,5-D-chiroIP₄, 1,2,4,6-D-chiroIP₄,1,2,5,6-D-chiroIP₄, 1,3,5,6-D-chiroIP₄, 1,4,5,6-D-chiroIP₄,2,3,4,5-D-chiroIP₄, 2,3,4,6-D-chiroIP₄, 2,3,5,6-chiroIP₄,2,4,5,6-D-chiroIP₄, 1,2,3,4,5-D-chiroIP₅, 1,2,3,4,6-D-chiroIP₅,1,2,3,5,6-D-chiroIP₅, 1,2,4,5,6-D-chiroIP₅, 1,3,4,5,6-D-chiroIP₄,2,3,4,5,6-D-chiroIP₅, and 1,2,3,4,5,6-D-chiroIP₆. In addition to thesephosphates, the invention also includes the corresponding pyrophosphateswhere at least one of the hydroxyl groups is phosphorylated by apyrophosphate rather than a monophosphate group, such as withoutlimitation compounds such as

which would be 3-pyrophosphatidyl D-chiroinositol. Any of the 6 hydroxypositions of the inositol ring can be substituted by a pyrophosphate andsome can be unphosphorylated or monophosphorylated with otherspyrophosphorylated or phosphorylated with higher phosphates.Non-limiting examples of mixed mono- and pyro phyosphorylatedD-chiroinositol include: the heptaphosphates such as1,2,3,4,5-pentamonophosphatidyl-6-pyrophosphatidyl-D-chiroinositol;1,2,3,4,6-pentamonophosphatidyl-5-pyrophosphatidyl-D-chiroinositol;1,2,3,5,6-pentamonophosphatidyl-4-pyrophosphatidyl-D-chiroinositol;1,2,4,5,6-pentamonophosphatidyl-3-pyrophosphatidyl-D-chiroinositol;1,3,4,5,6-pentamonophosphatidyl-2-pyrophosphatidyl-D-chiroinositol; and2,3,4,5,6-pentamonophosphatidyl-1-pyrophosphatidyl-D-chiroinositol; theoctaphosphates such as1,2-dipyrophosphotidyl-3,4,5,6-tetramonophosphatidyl-D-chiroinositol;1,3-dipyrophosphotidyl-2,4,5,6-tetramonophosphatidyl-D-chiroinositol;1,4-dipyrophosphotidyl-2,3,5,6-tetramonophosphatidyl-D-chiroinositol,1,5-dipyrophosphotidyl-2,3,4,6-tetramonophosphatidyl-D-chiroinositol;1,6-dipyrophosphotidyl-2,3,4,5-tetramonophosphatidyl-D-chiroinositol;2,3-dipyrophosphotidyl-1,4,5,6-tetramonophosphatidyl-D-chiroinositol;2,4-dipyrophosphotidyl-1,3,5,6-tetramonophosphatidyl-D-chiroinositol;2,5-dipyrophosphotidyl-1,3,4,6-tetramonophosphatidyl-D-chiroinositol;2,6-dipyrophosphotidyl-1,3,4,5-tetramonophosphatidyl-D-chiroinositol;3,4-dipyrophosphotidyl-1,2,4,5,6-tetramonophosphatidyl-D-chiroinositol;3,5-dipyrophosphotidyl-1,2,4,6-tetramonophosphatidyl-D-chiroinositol;3,6-dipyrophosphotidyl-1,2,4,5-tetramonophosphatidyl-D-chiroinositol;4,5-dipyrophosphotidyl-1,2,3,6-tetramonophosphatidyl-D-chiroinositol;and5,6-dipyrophosphotidyl-1,2,3,4,-tetramonophosphatidyl-D-chiroinositolamong others, for example another mixed octaphosphate, withoutlimitation is1-triphosphatidyl-3-pyrophosphatidyl-4,5,6-trimonophosphatidyl-D-chiro-inositol.For simplicity, a pyrophosphatidyl group will be indicated as “PP”, andlonger phosphate chains will be designated as “Poly(y)P”, where yindicates the number of phosphate groups in the chain, and y isgenerally not more than 4, but typically 3. Any of the free hydroxylgroups of the D-chiroinositol structure can be phosphorylated witheither a single phosphate group, a pyrophosphate group or a longerpolyphosphate chain of 3 or more phosphate groups and different hydroxylgroups in the same molecule can be phosphorylated with a variety of anyof a mono, di, or poly phosphate. Thus, for example, without limitation,1-monophosphatidyl-2-monopyrophosphatadiyl-D-chiroinositol is alsowithin the scope of the invention, as is1,2-di(monophosphatidyl)-3,4-diPP-5-Poly(3)P-D-chiroinositol,1,2,4,5,6-pentamonophosphatidyl-3-pyrophosphatidyl-D-chiroinositol, and1,2,5,6-tetramonophosphatidyl-3,4-dipyrophosphatidyl-D-chiroinositolalong with the corresponding compounds having a different distributionof the mono and pyro phosphate groups around the D-chiroinositol ring.When sterically possible, two hydroxyl groups of the D-chiroinositolstructure (within the same molecule can be linked together through asingle phosphate group, PP, or Poly(y)P group forming a ring structureor two or more D-chiroinositol molecules can be linked through suchphosphate groups as in the non-limiting structure III:

which exemplifies (but does not limit the invention to) a molecule inwhich two D-chiroinositol molecules are linked through a singlephosphate group between position 3 of one D-chiro-inositol and position1 of the other. The linking phosphate may be a single phosphate, a PP,or Poly(y)P group, and when two or more hydroxyl groups on the sameD-chiroinositol structure are phosphorylated, longer chains ofalternating D-chiroinositol and a phosphate (single phosphate, PP orPoly(y)P and mixtures thereof) are realized. Further, a phosphate or apyrophosphate may link two hydroxyl groups as for example, withoutlimitation, in structure IV below:

or in a more complex ring structure such as that of formula V below

or the two remaining phosphate hydroxyl groups can be dehydrates to forma P—O—P link as well. In addition, any of the six hydroxy groups thatare not phosphorylated can be substituted or replaced as indicatedelsewhere in this specification and any of the hydrogen atoms on the sixcarbons or the inositol ring may be replaced as indicated elsewhere inthis specification. Each of these more complex D-chiroinositolstructures are also within the scope of the present invention.Manufacture of the compounds having PP or Poly(y)P as thephosphorylating group (whether or not linking multiple D-chiroinositolunits together) can be prepared in an analogous fashion to the chemicalsynthesis of the phosphorylates that have only single phosphate groupsfor any one hydroxyl group by using pyrophosphate of Poly(y)P phosphatechains as the phosphorylation group source. With respect to the uses setforth herein other than in the prevention and treatment of spina bifidaand cancer treatments, the invention compounds also specifically includethe corresponding compounds, their phosphorylates and derivatives basedon the other inositol isomers as well. For example, of particularinterest to the inventor is the use or utility of the inositol higherphosphates and/or pyrophosphates to protect cells from alpha tumornecrosis factor (α-TNF) cell death as seen with autoimmune diseases andpathways involved described above. In analogous fashions to structuresIV and V, the —P(═O)(OH)— can be replaced by —S(O₂)— or —S(═O)—.

Formulations in the literature containing chiro-inositol,inositol-phosphates, etc., include, but are not limited to, thosedisclosed in U.S. Pat. Nos. 5,124,360; 5,614,510; 5,760,222; and6,784,209, all of which are incorporated herein by reference in theirentirety. Formulations of the D-chiro inositols of the invention andtheir phosphorylated, pyrophosphated, and polyphosphated derivatives asindicated as being useful in the present invention can be madeanalogously.

In each of the above D-chiroinositol structures and D-chiroinositolphosphate structures, further derivatives of the invention can be madeand utilized by replacement of one or more of the hydrogens on theD-chiroinositol ring or one or more of the hydrogens of one or more ofthe hydroxy groups on the D-chiroinositol ring or one or more of thereplaceable hydrogens on one or more of the phosphate groups shown instructures I-V above with a substituent selected form those indicatedearlier. In addition, one or more of the hydroxyl groups of the inositolring can be completely replaced with the “substituents” referred toearlier, except that if such hydroxyl group is completely replaced byhydrogen, then the other hydrogen of the same inositol ring carbon mustremain in place to be considered a D-chiroinositol derivative of thepresent invention (i.e. a deoxy variant), or both the hydrogen and thehydroxyl of the same carbon are replaced by a doubly bound oxygen (anoxo variant) or by a doubly bound nitrogen (an imino variant), while nosuch restriction generally exists if both the hydrogen and the hydroxylof the same carbon atom are replaced by substituents which are neitherhydrogen nor hydroxyl or derivitized hydroxy. For uses in which the baseinositol structure is not limited to the D-chiro inositol configuration,no such limitation on the replacements of the inositol ring hydroxylgroup exists, and all such allowable substituents within the abovesubstituent definitions are possible. Other replacements of the hydroxylgroup with additionally replacement of the other hydrogen of the sameinositol ring carbon retains the consideration of a D-chiroinositolderivative for purposes of the present invention unless specifically setforth otherwise. Preferable, non-limiting groups for the replacement ofone or more of these hydrogens on the D-chiroinositol ring carbonsinclude aliphatic groups, acylamino groups, alkoxy alkylamino,alkylthio, amino, aralkyl, carbonyl, derivitized carbonyl, thiocarbonyl,derivitized thiocarbonyl, and aryl to name a few, all of which may befurther unsubstituted or substituted in accordance with theaforementioned definitions of each of these terms. Other than thealkylthio and alkoxy, the same set of substituents is preferred forchoices to replace replaceable hydrogen of either the D-chiroinositolhydroxy groups or the replaceable hydrogens of the phosphate groups.Also, in any of the foregoing compounds having a D-chiroinositol ringfree hydroxy group, the hydroxyl group can be esterified or etherizedand one or more of the remaining hydrogens of the (6) six ring positioncan be replaced by an appropriate substituent as substituent is definedabove. Furthermore, One or more of the hydroxyl groups of the inositolring can be completely replaced by an appropriate substituent as definedabove. Suitable synthetic chemistry will be apparent to syntheticchemists once directed to a particular D-chiroinositol phosphate orderivative.

In other aspects of the invention, the present invention includes thecorresponding phosphorylated and/or pyrophosphorylated and/orpolyphosphorylated inositols and/or derivitized versions of suchinositols or derivitized versions of such phosphorylated,pyrophosphorylated, or polyphosphorylated inositols (which arederivitized in an analogous manner as that set forth for D-chiroinositolabove) where for purposes of this paragraph, “inositols” includes all ofthe inositol isomers for all of the foregoing indications other than theuse of myoinositol and its phosphorylates with phosphoric acid (mono- tohexa-monophosphates so that any one to all six of the inositol —OHgroups are replaced by a monophosphate) for prevention of spina bifidabirth defect and prevention or amelioration of breast cancer.

More specifically, and without limitation to the foregoing, compounds ofthe present invention (subject to the limitations of certain aspects tothose compounds based on the D-chiroinositol configuration and subjectto exclusion of certain compounds as set forth with respect to theirpresence in the prior art as set forth earlier), compounds for use inthe present invention include: (i)

wherein each of R₁₀₁, R₁₀₃, R₁₀₅, R₁₀₇, R₁₀₉, and R₁₁₁, is independentlyselected from H or a substituent R₂₀₁;

-   -   (a) each of R₁₀₂, R₁₀₄, R₁₀₆, R₁₀₈, R₁₁₀, and R₁₁₂, is        independently selected from OH, OR₂₀₂, OP(═O)(OR₂₁₁)(OR₂₁₂),        OP(═O)(OR₁₁₃)—OP(═O)(OR₂₁₁)(OR₂₁₂),        {OP(═O)(OR₁₁₃)—OP(═O)(OR₁₁₃)}_(a)—OP(═O)(OR₂₁₁)(OR₂₁₁)(OR₂₁₂)        (wherein a is 1-3); or a substituent R₂₀₃; or not more than 3 of        R₁₀₂, R₁₀₄, R₁₀₆, R₁₀₈, R₁₁₀, and R₁₁₂ is independently H; or    -   (b) both of the respective R groups on the same carbon (that is        independently pair R₁₀₁ and R₁₀₂, pair R₁₀₃ and R₁₀₄, pair R₁₀₅        and R₁₀₆, pair R₁₀₇ and R₁₀₈, pair R₁₀₉ and R₁₁₀, and pair R₁₁₁        and R₁₁₂) are together ═O or —N(R₂₀₄); or (ii)

where each of the groups R₁₁₁—R₁₁₂, in each unit are independently asset forth above except that one of such R groups in each of the terminalstructures is a direct bond to the indicated oxygen instead of theforegoing, and one of such R groups in each intermediary structure is adirect bond to one of the two indicated oxygens instead of the above anda second of the R groups in each intermediary structure is a direct bondto the other indicated oxygen, p, r, and s are each l, t and k are eachindependently an integer of from 0 to 2, and n is a an integer of from 0to 8; pharmaceutically acceptable salts thereof, and mixtures thereof,or in which in the structure above t=0 and any or all of the —P(O)(OH)—groups are independently replaced by —C(O)—,

-   -   —S(O)—, or —S(O)₂—;        wherein R₂₀₁, R₂₀₂, R₂₀₃, and R₂₀₄ are each independently        selected from halogen (preferably fluorine, chlorine, bromine,        or iodine, more preferably fluorine, or chlorine, most        preferably fluorine); trihalomethyl (preferably        trifluoromethyl), cyano, azido, unsubstituted or substituted        aliphatic groups, unsubstituted or substituted aromatic groups,        —C(O)—R₂₀₅, —C(O)—O—R₂₀₆, —S—C(O)—R₂₀₇, —C(O)—S—R₂₀₈,        —C(S)—R₂₀₅, —C(S)—O—R₂₀₆, —S—C(S)—R₂₀₇, —C(S)—S—R₂₀₈,        —SC(S)—S—R₂₀₈, —C(NR₂₀₉)—R₂₀₅, —C(NR₂₀₉)—O—R₂₀₆,        —S—C(NR₂₀₉)—R₂₀₇, —C(NR₂₀₉)—S—R₂₀₈, —S—R₂₁₀, —S(O)R₂₁₀,        —S(O)OR₂₁₀, —S(O)₂R₂₁₀, —S(O)₂OR₂₁₀, —NR₂₀₉R₂₁₀,        —N(R₂₀₉)—C(O)—O—R₂₀₆, —N(R₂₀₉)—C(O)—S—R₂₀₈, —N(R₂₀₉)—C(S)—R₂₀₅,        —N(R₂₀₉)—C(S)—O—R₂₀₆, —N(R₂₀₉)—C(S)—S—R₂₀₈,        —N(R₂₀₉)—C(NR₂₀₉)—R₂₀₅, —N(R₂₀₉)—C(NR₂₀₉)—O—R₂₀₆,        —N(R₂₀₉)—C(NR₂₀₉)—S—R₂₀₈, —S—R₂₁₀, —N(R₂₀₉)—S(O)R₂₁₀,        —N(R₂₀₉)S(O)OR₂₁₀, —N(R₂₀₉)S(O)₂O₂₁₀, —N(R₂₀₉)—S(O)₂O₂₁₀, and        —NR₂₀₉R₂₁₀, —P(O)(OR₂₁₁)(OR₂₁₂),        wherein each of R₂₀₅ through R₂₁₂ is itself independently        selected from H, an unsubstituted or substituted aliphatic        groups, or an unsubstituted or substituted aromatic groups,        wherein the aliphatic groups are selected from straight chain        and branched carbon chains, whether saturated or unsaturated, of        up to 30 carbon atoms, preferably of up to 20 carbon atoms, more        preferably of up to 10 carbon atoms, more preferably of up to 7        carbon atoms, most preferably of up to 5 carbons, especially        methyl, ethyl, propyl, and cycloaliphatic rings having 3-10 ring        members such rings being carbocyclic or heterocyclic where the        heterocyclic rings have one to four heteroatoms selected from        oxygen, sulfur, and nitrogen; the cycloaliphatic rings being        saturated or partially unsaturated, and the aromatic group        having 6-8 ring members selected from carbon, oxygen, sulfur,        and nitrogen, the aliphatic and aromatic groups further        containing up to four fused rings of either cycloaliphatic        rings, aromatic rings or both cyclosaliphatic and aromatic        rings, each of the aliphatic and aromatic groups being further        unsubstituted or substituted by hydroxy, C₁₋₇alkoxy, alkylthio,        C₁₋₂₀acyloxy, phosphate, halogen (preferably fluorine, chlorine,        bromine, or iodine, more preferably fluorine, or chlorine, most        preferably fluorine); trihalomethyl (preferably        trifluoromethyl), cyano, and azido; each substituent being mono        or multiply present as valence permits; and further provided        that in the foregoing substitution patterns, no substitution        pattern results in a peroxy group; the R₂₁₁ and/or the R₂₁₂ of        any —P(O)(OR₂₀)(OR₂₁₂) may be further joined to any free hydroxy        group or to result in phosphate containing rings such as for        example

or the corresponding compounds to structures (C), (D), and (E) wherein(a) the —P(OR₂₁₂)(O)—group is replaced by —C(O)—, —S(O)—, or —S(O)₂—orthe (OR₂₁₂) together with any remaining group R₁₁₁ through R₁₁₂ forms an—O— group resulting in additional fused rings; wherein each of R₂₀₅through R₂₁₃ is itself independently selected from H, unsubstituted orsubstituted aliphatic groups, and unsubstituted or substituted aromaticgroups, preferably,

and/or, the R₂₁₀ of any group —S(O)OR₂₁₀ or —S(O)₂R₂₁₀ may be joined toany free hydroxy group or to result in —S(O)— or —S(O)₂— containingrings analogous to structures IV and V except that the —P(O)(OH)— groupis replaced by —C(O)—, —S(O)— or —S(O)₂— respectively; or in which anyof the foregoing compounds having a free hydroxy group is esterifiedwith an acidic group of folic acid or an acidic group of apolysaccharide-folic acid compound, or any of the foregoing compoundshaving a free acidic group such as —P(═O)(OH), —C(═O)(OH), —S(═O)(OH),or —S(═O)₂(OH) is esterified with a free hydroxyl group of folic acid orof a polysaccharide-folic acid compound, or in which any of theforegoing having a free amine (—NHR (R being optionally H) or imine(═NH) forms together with the acidic group of a folic acid or of apolysaccharide-folic acid compound an amide (—C(═O)—NR—), a sulfonamide(—S(═O)₂—NR—), or a group —S(═O)—NR—.

Of particular interest are the multiple phosphorylated inositols having3 or more monophosphate groups per molecule without otherderivatizations, the IP₇, IP₈, and IP₉ inositols without otherderivatizations; the derivatized inositols where one or more of the ringhydrogens is replaced with a halogen, especially fluorine, an aminogroup, an azido group, or a cyano group; the compounds where one or moreof the inositol ring hydroxyl groups is replaced by a hydroxymethyl; andthose having the following substituents

where the alkyl is of 1-30 carbons and saturated or unsaturated,straight chain or branched, preferably octadecyloxy and the alkanoyl isup to 30 carbons and preferably saturated or unsaturated, straight chainor branched, preferably palmitoyl and the substituent is bound throughthe free oxygen atom to the inositol ring in place of one of theinositol ring hydroxyl groups or to an inositol analog having ahydroxymethyl group in place of another inositol ring hydroxyl group asfor example

with the particular position of the for the substituents on the inositolring and the particular orientation of the various hydroxyl groups notbeing critical. In other words the 2-methoxy-3-(palmitoyloxy oroctadecyloxy)-glycero phosphate substituent or the2-methoxy-3-(palmitoyloxy or octadecyoxy)-glycerocarbonate substituentcan be attached at any of the 6 ring positions and the hydroxymethylgroup can be at any of the inositol ring positions in place of ahydroxyl at that position. Also of importance are thedipalmitoylglycerophosphate esters of the various inositols other thanthat of myoinositol, i.e., those having the following substituent

bound through the free oxygen of the phosphate (shown at the left in thestructure above) to any of the inositol ring positions (and thecorresponding molecules in which the —P(O)(OH)— is replaced by one of—S(O)—, or —S(O)₂—) and those that have the dipalmitoylglycerocarbonateesters of the various inositols

Other important compounds of the invention include those having thegroup

bound through the free oxygen shown on the left of the above structureto any of the inositol ring positions. Where the compounds of structureVI has higher acyls (over 14 carbons) as the (C₁₋₃₀alkyl or C₁₋₃₀)groups and the linking group —P(═O)(OH)—, —C(═O)—, —S(O)—, or —S(O)₂— islimited to —P(═O)(OH)—, and there is one such group bound to theinositol ring, the compounds are phosphatidylinositols. Thesephosphatidylinositols being further substituted having such furthersubstitution in patterns in accordance with the present inventioncompounds are also valuable for as inhibitors for PDK1 and its pathways.

While not including the unmodified, unphosphorylated, underivitizedinositol isomers themselves in many embodiments, such isomers havingbeen derivitized and/or phosphorylated are included and includescyllo-inositol, epi-inositol, cis-inositol, allo-inositol,neo-inositol, muco-inositol, dextro-inositol, levo-inositol, andL-chiro-inositol phosphorylations/derivatizations. These materials maybe used in amounts of from below 2 mg/day to in excess of 8grams/kg/day. Any of the above inositols may be used in combination forthe inositol component of the invention and may be used with or withoutother active agents in an analogous fashion as described in more detailabove concerning D-chiroinositol. It should be noted that the hedgehog,patched, and GL1,2,3 gain-of function or loss of function can bedetermined for each of the above inositol based compounds with thescreening tests set forth in the examples. Without being limited totheory, those that have hedgehog gain-of-function or smoothenedloss-of-function can be used in an analogous manner to theD-chiroinositol uses set forth above and those that have hedgehogloss-of-function or smoothin gain-of-function are not for use in theprevention of birth defects purposes set forth above, but are of use ina number of the other indications of the present invention, especiallyin the prevention and treatment of various cancers and other indicationsreferred to hereinabove. Some particular compounds that are beyond theD-chiro inositol compounds described earlier are, without limitation,fluoroscylloinositol, fluoroepi-inositol, fluorocis-inositol,fluoroallo-inositol, fluoroneo-inositol, fluoromuco-inositol,fluorodextro-inositol, fluorolevo-inositol, fluoro D-chiro-inositol,deoxyscyllo-inositol, deoxyepi-inositol, deoxycis-inositol,deoxyallo-inositol, deoxynco-inositol, deoxymuco-inositol,deoxydextro-inositol, deoxylevo-inositol, deoxyD-chiro-inositol,aminoscylloinositol, aminoepi-inositol, aminocis-inositol,aminoallo-inositol, aminoneo-inositol, aminomuco-inositol,aminodextro-inositol, aminolevo-inositol, aminoD-chiro-inositol,ketoscyllo-inositol, ketoepi-inositol, ketocis-inositol,ketoallo-inositol, ketoneo-inositol, ketomuco-inositol,ketodextro-inositol, ketolevo-inositol, ketoD-chiro-inositol, sulfoscylloinositol, sulfo epi-inositol, sulfo cis-inositol, sulfoallo-inositol, sulfo neo-inositol, sulfo muco-inositol, sulfodextro-inositol, sulfo levo-inositol, sulfo D-chiro-inositol and furthersulfato phosphorylates of an inositol isomer where the inositol isomeris selected from the group consisting of scyllo-inositol, epi-inositol,cis-inositol, allo-inositol, neo-inositol, muco-inositol,dextro-inositol, levo-inositol, and D-chiro-inositol; and the inositolcompound having at least one phophsoryl group selected frommonophosphoryl groups, pyrophosphoryl, groups, and polyphosphory groupsand further having at least one sulfato group, which phosphoryl group,and/or sulfato group and/or remaining free hydroxyl groups are eachindependently unsubstituted or substituted with a suitable substituentas hereinbefore described. Further compounds for use in the inventionuses include D-chiro inositoll inositol derivative for metabolite isselected from the group of D-chiro-inositol phosphates, d-chiro-inositolesters, D-chiro-inositol ethers, D-chiro-inositol acetals,D-d-chiro-inositol ketals, polysaccharides containing D-chiro-inositols,and D-chiro-inositol phospholipids, which compounds are disclosed inU.S. Pat. No. 6,486,127, incorporated herein in its entirety byreference.

Inositol “salts” contemplated for use in the practice of all aspects ofthe present invention include any and all pharmaceutically acceptablesalts, which may be used in this aspect of the invention as well as theD-chiroinositol more specific aspects set for the earlier. Examples aresalts of therapeutically acceptable organic acids, such as acetic,fumaric, lactic, maleic, citric, malic, succinic, toluenesulfonic acid,and the like, salts of polymeric acids, such as tannic acid, alginicacid, carboxymethylcellulose, and the like, and salts of inorganicacids, such as hydrochloric acid, sulfuric acid, and the like. Others,however, may also be utilized. Inositol “derivatives” employed in thepractice of this aspect of the present invention (as well as those setforth concerning the D-chiroinositol specific aspects set forth earlier)include those which have been modified as set forth earlier as well asthose modified to vary the hydrophilic or lipophilic character of theinositol molecules. Such modifications may be desirable to tailor thesolubility characteristics of the inositols to a particular mode orroute of administration. For example, lipophilic side chains, includingC₁₋₂₀ hydrocarbon chains, which may be saturated or unsaturated andcontain one or more non-hydrophilic substituents, may be added, as wellas conjugating the inositols to a lipophilic molecule to enhance itslipid solubility. Alternatively, the addition of hydrophilic side chainsor inositols conjugates to a hydrophilic molecule will enhance thehydrophilicity of the inositols, including C₁₋₂₀ hydrocarbon chainswhich may be unsaturated, and may have hydrophilic substituents such asHO, HS, NH₂, halo, keto, and the like. Exemplary inositol derivativescontemplated for use in the practice of one aspect of the presentinvention include amine-substituted, halogen-substituted, deoxy-, keto-,and sulfo-inositol analogues, and the like, as well as combinations ofany two or more substituents thereof. Preferred inositol derivativesinclude substituted inositol derivatives, including hydroxy, amino,halo, e.g., fluoro, deoxy, keto, and sulfo inositol analogues, amongother hydrophilic substituents, and combinations thereof, as well as thecorresponding salts. In some embodiments of the invention, the furthersubstituents set forth earlier in this specification are useful as well.

C. Exemplary Applications of Methods and Compositions

Folic acid (C₁₉H₁₉N₇O₆) and folates are well known in the art as arevarious formulations the ref. Any of the recognized folates orC₁₉H₁₉N₇O₆ is suitable for use in the present invention embodiments thatinclude a folic acid and/or folate component.

Women of child bearing age frequently are avoiding pregnancy byutilizing birth control pills. These are typically estrogenic substancesthat are administered for a time period and then either stopped for ashort time, continued at altered dosage, and/or supplemented or replacedby progestogenic substances so as to induce menses. During the timeframe when the estrogenic substance is reduced or stopped, it ispossible for a woman to become pregnant. On occasion, it is alsopossible that the intended “birth control” function of the birth controlpills (even when containing a full complement of the estrogenicsubstance) may not be totally efficacious, such as when othermedications or other substances are ingested that interfere with theproper workings of the birth control medication. In such situations apregnancy may result despite being on such medications. Although thereis a general awareness among pregnant women to have propersupplementation with folic acid, many women taking birth controlmedication do not take adequate supplements of folic acid or many othernutrients that are important to fetal development, simply because theybelieve that do not need to be concerned with a pregnancy at that time.Others are simply unaware of the need for adequate supplementation, andstill others, even though educated about this either neglect to takeappropriate supplements or still don't care. Others do not botherbecause of economic reasons. One aspect of the present invention is toinclude supplemental D-chiroinositol (and/or one of its phosphorylatedderivatives) into birth control pills which may further have folic acid(or other appropriate folate source) incorporated into some or all ofthe pills in the birth control pill package so as to assure that thewoman taken such birth control has adequate stores of D-chiroinositol(and folate, when folate is also incorporated) in the event that shebecomes pregnant either while taking birth control pills or during thetime period when she initially stops the birth control pill regimen.This is extremely important since both D-chiroinositol and folate aremost effective against the various fetal defects that the presentinvention is directed toward preventing when these substances areadministered pre-conception through the first trimester of pregnancy.The D-chiroinositol (and phosphorylated derivatives) and folic acid (andother oblates) can be incorporated into just the tablets of the birthcontrol pill package that have either no other active or haveprogestogenic but not estrogenic substances or have progesterins and lowlevels of estrogens present, but preferably are incorporated into all ofthe tablets. This is suitable because generally the higher estrogenicsubstance tablets will prevent pregnancy and the remaining tablets willbegin administering folic acid and D-chiroinositol (or theircounterparts) with the first tablet after the estrogenic tablets.However, it is preferable to have the compounds of the invention in allof the tablets in case of a pregnancy that results from birth controltablet failure or due to interference with proper action of theestrogenic substance due to drug interactions or other dietary orenvironmental impacts that cause birth control failure.

Another aspect of the invention is a combination product having bothD-chiroinositol (and/or a phosphorylated (either P, PP, and/or polyP)derivative thereof and/or other derivative thereof as set forth above)(hereinafter all such compounds collectively referred to, whether assingle agents or combinations of these agents, as “the D-chiroinositolcompound”), and folic acid (and/or other folate source) in a singlecomposition as a supplement that is especially suited for women of childbearing age who are not yet pregnant (but generally intending to becomepregnant), women who are not pregnant and not intentionally trying toobecome pregnant, but may be, and women who are pregnant. Such fixedcombinations may be a standalone product or have other nutritionalsupplements (or other active agent) incorporated therein. Suchadditional supplements include vitamins and minerals as well as herbalproducts and are well known (both as to substances and their respectivedosages) to those of ordinary skill in the nutritional supplement area.Without being held to theory, it is the inventors belief andunderstanding that co-therapy of C₁₉H₁₉N₇O₆ (and/or other folatesources) together with D-chiroinositol (and/or phosphorylatedderivatives (P, PP and/or polyP) thereof and/or other derivative thereofas set forth above), whether simultaneously or sequentially, operate ina manner that provides the best protective effects against fetalmalformations beyond those achievable with either component alone, andfurther that such results are better than those achieved with each aloneor that would have been predicted as additive effect. As such, suchco-therapy is also within the scope of the present invention, whethersuch co-therapy is via a fixed combination C₁₉H₁₉N₇O₆ (and/or otherfolate) and D-chiroinositol (and/or phosphorylated derivates (P, PPand/or polyP) thereof and/or other derivative thereof as set forthabove) or via separate administration of these agents generally within12 hours of each other and generally on a daily basis. Fractional dosingof either or both components taken multiple times a day (i.e., forexample ½ daily doses taken twice daily or ⅓ daily dosing taken threetimes daily) is also within the scope of the present invention.Fractional dosing multiple times a day is particularly suitable when thecomposition contains only nutritional supplements as active agents andwhen the patient finds that singe daily doing upsets the stomach or thedaily dose is large and not suitable for inclusion into a single unitdosage form.

An additional benefit of administering D-chiroinositol to women onestrogenic medications is downregulating the estrogen-receptor and/or,ErbB receptor overexpressor phenotypes and proliferation from estrogenicinsult. For example, D-chiro and/or its derivatives are likely mediatedby the production of second messenger lipids that elicit transmemebranesignal transduction cascades governing the activation and inhibition ofdownstream effectors. These views are also in line with the ideas onnon-estrogen receptor associated actions of the compound by way ofpromoting binding sites that govern cellular proliferation. Thus,incorporation of D-chiroinositol (and/or its phosphorylated (P, PPand/or polyP) derivatives and/or other derivative thereof as set forthabove) into fixed combinations with estrogenic medications is a means toincrease the safety of the use of estrogenic substances. WhileD-chiroinositol (and/or its phosphorylated (P, PP and/or polyP)derivatives and/or other derivative thereof as set forth above) can beused as separate medications or supplements in co-therapy with theestrogenic medication, it is highly preferred to have theD-chiroinositol compound as a fixed combination with the estrogenicsubstance as to assure patient compliance. While estrogenic sensitivebreast tissue in men is rarer than in women, it does occur andco-therapy in men having estrogenic treatment is also within the scopeof the present invention. Furthermore, since estrogenic insult is theresult of excess estrogen from endogenous overproduction of estrogen,exogenous administration of estrogen, insufficient androgenicproduction, or exogenous administration of anti-androgens (androgenablative therapy), the present invention also includes treating men orwomen with co-therapy of D-chiroinositol compound with anti-androgens,which co-therapy can be by separate administration of the compounds ofthe invention with such anti-androgens or via fixed combinationstherewith. The invention still further includes treating patients withconditions that result in excess estrogen or conditions that result inestrogenic-receptor overexpression phenotypes (whether because ofoverproduction of estrogen or insufficient androgen production, orcongenital deformation in the breast architecture microenvironment) withthe D-chiroinositol compound as a means of decreasing the risk of breastcancer from excess estrogenic insult. Finally, in this group oftreatments of the invention, the invention further includes treatingpatients with a general overproduction of hormonal steroids (even thoughestrogenic/androgenic balance is maintained). A still further benefit towomen who are or become pregnant while receiving the present inventiontreatment is that of reducing the incidents of gestational diabetes (orif they still do have such, it is a milder case), especially since therehas been a connection between gestational diabetes and some fetalmalformations due poor maternal phosphoinositide turnover orderangement. Specific active agents which can be combined for co-therapywith D-chiroinositol compound and/or derivatives and optionally withaddition folic acid (or other folate source) that are within theinvention include, without limitation: antiprogestogens, androgens,antiandrogens, estrogens, selective estrogen receptor modulators,aromatase inhibitors, gonadotropins, ovulation stimulators, gonadotropinreleasing hormone agonists, gonadotropin releasing hormone antagonists,LHRH agonists, progestins, and anti-progestins, to name a few. Many ofthese classes are utilized in opposing conditions but the co-therapywith the D-chiroinositol component of the invention and optionally thefolate component of the invention is warranted in that in some cases,the effect of the D-chiroinositol component (and optional folatecomponent) is complementary to the other active agent, while in othercases, the D-chiroinositol component (and optional folate component) areprotective of one or more of the potential side effects of the otheractive agent. Specific compounds belonging to these classes of otheractive agents are exemplified in the following non-exclusive,non-limiting list, each agent of which is prepared in its normal knownmethod and utilized in its known dosage, and include without limitation:13-cis-Retinoic Acid, 2-CdA, 2-Chlorodeoxyadenosine, 5-Azacitidine,5-Fluorouracil, 5-FU, 6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine,Abraxane, Accutane, Actinomycin-D, Adriamycin®, Adrucil®, Agrylin®,Ala-Cort®, Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ®,Alkeran®, All-transretinoic Acid, Alpha Interferon, Altretamine,Amethopterin, Amifostine, Aminoglutethimide, Anagrelide, Anandron®,Anastrozole, Arabinosylcytosine, Ara-C, Aranesp®, Aredia®, Arimidex®,Aromasin®, Arranon®Arsenic Trioxide, Asparaginase, ATRA Avastin®,Azacitidine, BCG, BCNU, Bevacizumab, Bexarotene, BEXXAR®, Bicalutamide,BiCNU, Blenoxane®, Bleomycin, Bortezomib, Busulfan, Busulfex®, C225,Calcium Leucovorin, Campath®, Camptosar®, Camptothecin-11, CapecitabineCarac™, Carboplatin, Carmustine, Carmustine, Wafer Casodex®, CC-5013,CCNU, CDDP, CeeNU, Cerubidine®, Cetuximab, Chlorambucil, Cisplatin,Citrovorum Factor, Cladribine, Cortisone, Cosmegen®, CPT-11,Cyclophosphamide, Cytadren®, Cytarabine, Cytarabine LiposomalCytosar-U,®Cytoxan®, Dacarbazine, Dacogen, Dactinomycin, DarbepoetinAlfa, Dasatinib, Daunomycin, Daunorubicin, Daunorubicin Hydrochloride,Daunorubicin Liposomal, DaunoXome®, Decadron, Decitabine, Delta-Cortef®,Deltasone®, Denileukin, diftitox, DepoCyt™, Dexamethasone, Dexamethasoneacetate, Dexamnethasone Sodium Phosphate, Dexasone, Dexrazoxane, DHAD,DIC, Diodex, Docetaxel, Doxil®, Doxorubicin, Doxorubicin liposomalDroxia™, DTIC, DTIC-Dome®, Duralone®, Efudex®, Eligard™, Ellence™,Eloxatin™, Elspar®, Emcyt®, Epirubicin, Epoetin alfa, Erbitux™,Erlotinib, Erwinia, Etanercept, L-asparaginase, Estramustine, Ethyol,Etopophos®, Etoposide, Etoposide Phosphate, Eulexin®, Evista®,Exemstanc, Farcston®, Faslodex®, Femara®, Filgrastim, Floxuridine,Fludara®, Fludarabine, Fluoroplex®, Fluorouracil, Fluorouracil (cream),Fluoxymnesterone, Flutamide, Folinic Acid, FUDR®, Fulvestrant, G-CSF,Gefitinib, Gemcitabine, Gemtuzumnab, ozogamicin, Gemozar®, Gleevec™,Gliadel®Wafer, GM-CSF, Goserelin, Granulocyte—Colony Stimulating Factor,Granulocyte Macrophage Colony Stimulating Factor, Halotestin®,Herceptin®, Hexadro, Hexalen®, Hexamethylmelamine, HMM, Hycamtin®,Hydrea®, Hydrocort Acetate®, Hydrocortisone, Hydrocortisone SodiumPhosphate, Hydrocortisone Sodium Succinate, Hydrocortone Phosphate,Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin®, Idarubicin,Ifex®, IFN-alpha 1 fosfamide, IL-11, IL-2, Imatinib mesylate, ImidazoleCarboxamide, Interferon alfa, Interferon Alfa-2b (PEG Conjugate),Interleukin-2, Interleukin-11, Intron A®(interferon alfa-2b), Iressa®,Irinotecan, isotretinoin, Kidrolase®, Lanacort®, Lapatinib,L-asparaginase, LCR, Lenalidomide, Letrozole, Leucovorin, Leukeran,Leukine™, Leuprolide, Leurocristine, Leustatin™ Liposomal, Ara-C LiquidPred®, Lomustine, L-PAM, L-Sarcolysin, Lupron®, Lupron Depot®,Matulane®, Maxidex, Mechlorethamine, Mechlorethamine Hydrochloride,Medralone®, Medrol®, Megace®, Megestrol, Megestrol Acetate, Melphatan,Mercaptopurine, Mesna, Mesnex™, Methotrexate, Methotrexate Sodium,Methylprednisolone, Meticorten®, Mitomycin, Mitomycin-C, Mitoxantrone,M-Prednisol®MTC, MTX, Mustargen®, Mustine, Mutamycin®, Myleran®,Mylocel™, Mylotarg®, Navelbine®, Nelarabine, Neosar®, Neulasta™,Neumega®, Neupogen®, Nexavar®, Nilandron®, Nilutamide, Nipent®, NitrogenMustard, Novaldex®, Novantrone®, Octreotide, Octreotide acetate,Oncospar®, Oncovin®, Ontak®, Onxal™, Oprevelkin, Orapred®, Orasone®,Oxaliplatin, Paclitaxel, Paclitaxel Protein-bound, Pamidronate,Panitumumnab, Panretin®, Paraplatin®, Pediapred®, PEG Interferon,Pcgaspargase, Pegfilgrastim, PEG-INTRON™, PEG-L-asparaginase,PEMETREXED, Pentostatin, Phenylalanine Mustard, Platinol®, Platinol-AQ®,Prednisolone, Prednisone, Prelone®, Procarbazine, PROCRIT®, Proleukin®,Prolifeprospan 20 with Carmustine Implant, Purinethol®, Raloxifene,Revlimnid®, Rheumatrex®, Rituxan®, Rituximab, Roferon-A®(InterferonAlfa-2a), Rubex®, Rubidomycin hydrochloride, Sanidostatin®, SandostatinLAR®, Sargramostim, Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™,STI-571, Streptozocin, SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®,Targretin®, Taxol®, Taxotere®, Temiodar®, Temazolomide, Teniposide,TESPA, Thalidomide, Thalomid®, TheraCys®, Thioguanine, ThioguanineTabloid®, Thiophosphoamide, Thioplex®, Thiotepa, TICE®, Toposar®,Topotecan, Toremifene, Tositumomab, Trastuzumnab, Tretinoin, Trexall™,Trisenox®, TSPA, TYKERB®, VCR, Vectibix™, Velban®, Velcade®VePesid®,Vesanioid®Viadur™, Vidaza®, Vinblastine, Vinblastine Sulfate, VincasarPfs®, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VM-26,Vorinostat, VP-16, Vumon®, Xeloda®, Zanosar®, Zevalin™, Zinecard®,Zoladex®, Zoledronic acid, Zolinza, Zometa®, etc. A preferred setincludes, without limitation: abarelix, abraxane (paclitaxel),adriamycin (doxorubicin), algestone, amadinone, aminoglutethimide,anagestrone, anastrozole, androisoxazole, androstanolone,androstenediol, 4-androstene-3,16,17-trione, aredia (pamidronatedisodium), arimidex (anastrozole), aromasin(exemestane), bazedoxifene,benorterone, bicalutamide, bolandiol, bolasterone, bolazine, boldenone,bolenol, bolmantalate, buserelin, calusterone, chemotherapy regimens,(cyclophosphamide (cytoxan), methotrexate (amethopetrin, Mexate, folex,and fluororucil (fluorourcil, 5-fu, adrucil) (this therapy is calledCMF), cyclophospamide, doxorubicin (adriamycin) and fluorouracil (thistherapy is called CAF), doxorubicin (adriamycin) and cyclophosphamide(this therapy is called AC), doxorubicin (adriamycin) andcyclophosphamide with paclitaxel (taxol), doxorubicin (adriamycin)followed by CMF, cyclophosphamide, eprubicin9ellence), and fluororacil,chlorotrianisene, chorionic gonadotropin, cioteronel, cingestol,clogestone, clomegestone, clometherone, clomifene, clostebol, conjugatedestrogens, cyproterone, cytoxan (cyclophasphamide), danazol,delmiadinone, deslorelin, desogestrcl, detirelix, dienestrol,diethylstilbestrol, dimethisterone, dihydrogestrone, drospirenone,drostanolone, dydrogesterone, ellence (epirubicin), epiestriol,epimestrol, epitiostanol, epristeride, equilin, esterified estrogens,estradiol, estrazinol, estriol, estrofurate, estrone, estropipate,ethinylestradiol, ethisterone, ethylestrenol, ethynerone, ethynodiol,etonogestrel, evista (raloxifene), exemestane, fareston (toremifene),femara (letrozole), fenestrel, finasteride, fluoxymesterone,flurogestone, flutamide, formebolone, formestane, fosfestrol,fulvestrant, furazabol, ganirelin, gestaclone, gestadienol, gestodene,gestonorone (especially gestonorone caproate), gestrinone, gonadorelin,goserelin, haloprogesterone, herceptin (trastuzumab), histrelin,4-hydroxy-19-nortestosterone, hydroxyprogesterone, ibutamoren,idoxifene, infliamab, letrozole, leuprolide, leuprorelin,levonorgestrel, lutrelin, lynestrenol, mebolazine, medrogestone,medroxyprogesterone, megace (megestrol), melengestrel, menotropins(especially humegon, pergonal, repronex), mesabolone, mestranol,mesterolone, metandienone, metenolone, methandriol, methenolone,methestrol, methyltestosterone, methynodiol, metribolone, mibolerone,mifepristone, nafarelin, nafoxidine, nandrolone, nilutamide,nitromifene, norboletone, norbolethone, norclostebol, norelgestromin,norethandrolone, norethindrone, norethisterone, norethynodrel,norgestimate, norgestomet, norgestrel, norgestrienone, nylestriol,oxabolone, oxandrolone, oxendolone, oxogestone, oxymesterone,oxymetholone, polyestradiol (especially polyestradiol phosphate),pralmorelin, prasterone, progesterone, quinbolone, quinestrol,quinestradol, quingestanol (especially quingestanol acetate),quingestrone, raloxifene, rismorelin, somalapor, somatrem, somatropin,somenopor, somidobove, stanozolol, stenbolone, sumorelin, tamoxifen,taxol (palitaxel), taxotere (docetaxel), testosterone, tibolone,tigestrol, tiomesterone, topterone, toremifene, trenbolone,trimegestone, trioxifene, triptorelin, urofollitropin, vorozole, xeloda(capecitabine), zanoterone, and zeranol, zoladex (goserelin), zometa(zoledronic) among others, each of which includes the pharmaceuticallyacceptable salts and esters thereof. These are all known compounds withknown uses and are used in the normal course for those knownindications. The co-therapy of the present invention adds theD-chiroinositol compound and optionally folic acid (and/or other folatesource) thereto, with the amounts of the D-chiroinositol components andfolic acid components being as set forth elsewhere herein. TheD-chiroinositol and optional folate can be separately administered withthese other active agents of combined in fixed combinations therewith asmay be convenient.

Turning to the fetal malformations of the present invention, fetaldevelopment is a very delicate and sensitive process and there are manypoints at which something can go wrong, resulting in a congenitaldefect. As such, no treatment will eliminate all such fetal defects oreven all occurrences of any one type of fetal defect. Nonetheless, theadministration of D-chiroinositol (and/or phosphorylated derivatives (P,PP and/or polyP) thereof) alone or in combination with folic acid(and/or other folate source) during the first trimester of pregnancy,preferably throughout the first trimester of pregnancy, even morepreferably from before conception into the first trimester of pregnancy,and most preferably from before conception through at least the end ofthe first trimester of pregnancy will significantly reduce the frequencyof a wide range of fetal defects, above those reported previously forthose patients who have not been treated or those patients who have beentreated with either of the D-chiroinositol (and/or its phosphorylated(P, PP and/or polyP) derivatives) or with folic acid (or other folatesource) alone (where those treatments have been previously studied. Thetreatment of the present invention further reduces the frequency ofthese defects as compared to treatment with other forms of inositol(and/or phosphorylated derivatives thereof) where such treatment hasbeen previously studied.

The defects, the frequency of which the present invention is designed toreduce, include, but are not limited to wherein the defect isVATER/VACTERL association (vertebral [defects], [imperforate] anus,tracheoesophageal [fistula], radial and renal [dysplasia])rachischisis(aka spinal dysraphism) such as spina bifida (including, but not limitedto spina bifida aperta (aka spinabifida cystica); spinabifida occulta;and occult spinal disorder, among others) and (b) craniorachischisis(aka cranial dysraphism) such as cranium bifida (aka encephalocele orcraniocele) each of spina bifida and cranium bifida being of any of thefollowing types meningocele, myelomeningocele, lipomeningocele, andlipomyclomcningocele among others; (c) anencephaly; and (d) chiarimalformation types 0,12,3; (2) caudal regression syndrome, caudaldysplasia sequence, congenitalsacral agenesis; sironmelia (mermaidsyndrome), sacral regression and the like; (3) cranio-facial defectssuch as, without limitation, facial cleft (aka prosopoanoschisis,including without limitation cleft palate, cleft lip, velopharyngealmalformation (including without limitation bifid uvula), etc.); (4)anorectal malformations including, but not limited to (a) imperforateanus, (b) rectoperineal fistula, (c) recto-bladder neck fistula; (d)persistent urogenital sinus, (e) persistent cloaca, etc.; (5)bucket-handle malformation; among others. Biemond syndrome, Chiarimalformation, (0,1,2,3), Ectrodactyly-ectoderma dysplasia, cleftlip/palate, Ellis Van Creveld syndrome, Muir-Torre syndrome, Cowdensyndrome, Carney complex, Birt-Hogg-Dubé syndrome, Gorlin syndrome (ptcloss-of-function), Gorlin-Goltz syndrome, basal cell nevus syndrome,bifid-rib basal-cell nevus syndrome, basal cell cancer syndrome (shhgain of function), and multiple basal cell nevi, squamous cell carcinoma(increased ptc activity) Meckel Gruger syndrome,McKusick-Kaufmansyndrome, Mirror hand deformity (ulnar dimelia) Mohrsyndrome, Oral-facial-digital syndrome, Pallister Hall syndrome,cephalopolysyndactyl), Post axial polydactyly, GreigRubinstein-Taybisyndrome, retinoblastoma, Cardiofaciocutaneous syndrome, Noonansyndrome, short rib polydactyly, extra deformed fingers and toes, Lowesyndrome including ocular and renal defects, Renal Colombo syndrome,retinoblastoma, retinitis pigmentosa, holoprosencephaly, maculardegeneration (whether it be due to a Shh defects, age, or secondaryconditions like diabetes mellitus), mental retardation. All of theseterms are well known in the art. However, for rapid reference, thoseunfamiliar with these terms are referred (without limitation to theMerck Manual, Eighteenth Edition 2006 and the PDR Medical Dictionary,Second Edition, 2000. The ultimate cause of these conditions can begenetic or environmental, or both. Nonetheless, it is also postulatedhere that certain cancers, and possibly even breast cancer in offspringare the result of signaling defects in utero and should be considered abirth defect as well since the results of the signaling defects in uteromay not present until much later in life similar to that of othercancers when DNA damage and mutations accumulate postnatally. Forexample, our research suggests that in the microenvironment of mammarybreast architecture comprise a population of epithelial cells and stemcells that continue to communicate postnatally through convergedsignaling pathways. These mammary epithelial cells have the ability forself-renewal and harbor tumorigenic potential. Subsequent alterations inpostnatal signaling mechanisms can result in a breast cancer assubsequent mammary development proceeds postnatally. Further studiesalso suggest that the stem-like, self-renewing cells originate from theprogenitor fetal cells during early embryonic mammary development. Thesebreast cancer stem cells have been identified as CD44⁺CD24⁻ breast tumorcells. In certain mouse mammary experimental models stem/progenitorcells displayed sensitivity to altered or hyper states of signalingpathways and new mapping techniques have demonstrated that a populationof adult neural stem cells that rarely divides responded to normal orhyperactive Shh signaling even though adult neural stem cells rarelydivide. All of this could result in unintended consequences in theformation of new malignancies. Nature 437, 894-897(6 Oct. 2005). Wepropose to correct signaling defects in utero with the method compoundand/or its derivatives to prevent breast cancer.

Without being bound to theory, the inventor believes that all of theseconditions are related to failure of proper embryonic patterning(mapping sequences) during the critical embryonic first trimester. Oneembryonic patterning sequence that has been identified is the Sonichedgehog (Shh) gene and some inositol phosphates (and kinases therefore)(PI3K) have been shown to be important in the proper expression of theShh gene. It is the present inventor's belief that these two signalingpathways converge in order for sufficient gene expression to occur.Mutations in the human shh gene and genes that encode its downstreamintracellular signaling pathway causes many clinical disordersincluding, but no way limited to basal cell carcinoma, nevoid basal cellcarcinoma syndromes along with distinct congenital syndromes syndromesas described above. Thus, insufficient D-chiroinositol levels (and/orphosphates (P, PP and/or polyP) thereof) interfere with or prevent theproper expression of the Shh gene and the result thereof is impropersignaling of proper mapping of the embryonic tissue. Thus, propersupplementation with the D-chiroinositol compound will restore propersignaling and mapping, second messenger systems involved in embryonicpatterning at that critical period, especially, if the embryo is one atrisk of such improper signaling and mapping) so as to substantiallyreduce and/or eliminate the risk of the presentation of the above fetalmalformations. Since the risk of some of the above conditions have beenshown (but not statistically significant) to benefit from folatesupplementation, co-therapy with both D-chiroinositol (and/or itsphosphorylated (P, PP and/or polyP) derivatives) and folic acid (and/oranother folate source) is the preferred embodiment of the invention. Itis also believed that the D-chiroinositol (plus one of itsphosphorylated (P, PP and/or polyP) derivatives) is the natural activeagent involved in this mechanism and that either other forms likemyo-inositol have a weak (or weaker) effect alone. It is furtherbelieved that a significant number of the women having children withthese malformations have (a) insufficient inositol intake and thereforecannot convert a sufficient amount to the D-chiro form or (b) simplycannot properly convert other inositol forms to the D-chiro varietyand/or to the proper phosphorylated variety via phosphoinositides. Inthis subpopulation, supplementation with any of the D-chiroinositoland/or its phosphorylated derivatives will serve equally well. A smallsubpopulation however may have defects in the various kinases (forexample, PKA or PKC isoforms) involved and thus, the bestsupplementation would be with the particular phosphorylate that is afterthe kinase defect. Since finding the specific defect in a particularkinase may not be easily identified in all cases, a separate embodimentof the present invention is to use a mixture of D-chiroinositol and anumber of its phosphorylated (P, PP and/or polyP) derivatives so as tobe sure that none of the advantages of the present invention are missedin as many patients as possible. A highly preferred embodiment in thiscase is to use a mixture of D-chiroinositol and at least one memberselected from D-chiroinositol-Phosphates₍₁₋₈₎. (InD-chiroinositol-Phosphates₍₂₋₈₎, one or more of the phosphates may be inthe form of pyrophosphates, and in D-chiroinositol-Phosphates₍₇₋₈₎ atleast one of the phosphates must be present as a pyrophosphate as thereare only 6 positions which can be monophosphorylated).

Another aspect of the present invention relates to a method ofmodulating a differentiated state, survival, and/or proliferation of acell, such as a normal cell or a cell having a ptc loss-of-function,hedgehog gain-of-function, or smoothened gain-of-function, or by anaberrant PI3K signaling pathway by contacting the cells with a compoundas set forth above according to the subject method and as thecircumstances may warrant as a way to target, manipulate, to optimizecontrol of this pathway thereby mitigating its contribution to oncogenicand embryonic patterning activity.

Despite significant progress in reconstructive surgical techniques,scarring can be an important obstacle in regaining normal function andappearance of healed skin. This is particularly true when pathologicscarring such as keloids or hypertrophic scars of the hands or facecauses functional disability or physical deformity. In the severestcircumstances, such scarring may precipitate psychosocial distress and alife of economic deprivation. Wound repair includes the stages ofhemostasis, inflammation, proliferation, and remodeling. Theproliferative stage involves multiplication of fibroblasts andendothelial and epithelial cells. Through the use of the subject method,the rate of proliferation of epithelial cells in and proximal to thewound can be controlled in order to accelerate closure of the woundand/or minimize the formation of “scar tissue”. Fibroblasts can bestimulated Myo-ip6-SO₄ (Stabilizers for fibroblast growth factors,Middaugh et al, U.S. Pat. No. 5,348,941) and play a critical role inwound healing. Transplanted fibroblasts can often retain positionalmemory of the location and tissue context where they had previouslyresided, at least over a few generations. Thus, the present inventionfinds utility in joints, hip, knee, cell, and tissue replacement. In oneembodiment, embedding a fibroblast with the compound D-chiro-Ip6-SO₄ ina depot formulation, into the surgical site to help wound healing willpromote the wound healing, especially in an elderly population that doesnot heal well due to age or people with diabetes mellitus, or immunesystem problems etc.

For instance, it is contemplated by the invention that, in light of thefindings of an apparently broad involvement of PI3K and hedgehog, ptc,and smoothened in the formation of ordered spatial arrangements ofdifferentiated tissues in vertebrates, the subject method is suitablefor use as part of a process for generating and/or maintaining an arrayof different vertebrate tissue both in vitro and in vivo. The compound,whether inductive or anti-inductive with respect proliferation ordifferentiation of a given tissue, can be, as appropriate, any of thepreparations described above.

For example, the present method of using subject compounds is applicableto cell culture techniques wherein it is desirable to control theproliferation or differentiation of the cell. A subject compound may beemployed in a method directed towards cells which have a ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function phenotype. In vitro neuronal culture systems haveproved to be fundamental and indispensable tools for the study of neuraldevelopment, as well as the identification of neurotrophic factors suchas nerve growth factor (NGF), ciliary trophic factors (CNTF), and brainderived neurotrophic factor (BDNF). One use of the present method may bein cultures of neuronal stem cells, such as in the use of such culturesfor the generation of new neurons and glia. In such embodiments of thesubject method, the cultured cells can be contacted with a compound ofthe present invention in order to alter the rate of proliferation ofneuronal stem cells in the culture and/or alter the rate ofdifferentiation, or to maintain the integrity of a culture of certainterminally differentiated neuronal cells. In an exemplary embodiment,the subject method can be used to culture, for example, sensory neuronsor, alternatively, motorneurons. Such neuronal cultures can be used asconvenient assay systems as well as sources of implantable cells fortherapeutic treatments.

According to the present invention, large numbers of non-tumorigenicneural progenitor cells can be perpetuated in vitro and their rate ofproliferation and/or differentiation can be affected by contact withcompounds of the present invention. Generally, a method is providedcomprising the steps of isolating neural progenitor cells from ananimal, perpetuating these cells in vitro or in vivo, preferably in thepresence of growth factors, and regulating the differentiation of thesecells into particular neural phenotypes, e.g., neurons and glia, bycontacting the cells with a subject compound.

Progenitor cells are thought to be under a tonic inhibitory influencewhich maintains the progenitors in a suppressed state until theirdifferentiation is required. However, recent techniques have beenprovided which permit these cells to be proliferated, and unlike neuronswhich are terminally differentiated and therefore non-dividing, they canbe produced in unlimited number and are highly suitable fortransplantation into heterologous and autologous hosts withneurodegenerative diseases.

By “progenitor” it is meant an oligopotent or multipotent stem cellwhich is able to divide without limit and, under specific conditions,can produce daughter cells which terminally differentiate such as intoneurons and glia. These cells can be used for transplantation into aheterologous or autologous host. By heterologous is meant a host otherthan the animal from which the progenitor cells were originally derived.By autologous is meant the identical host from which the cells wereoriginally derived.

Cells can be obtained from embryonic, post-natal, juvenile or adultneural tissue from any animal. By any animal is meant any multicellularanimal which contains nervous tissue. More particularly, is meant anyfish, reptile, bird, amphibian or mammal and the like. The mostpreferable donors are mammals, especially mice and humans.

In the case of a heterologous donor animal, the animal may beeuthanized, and the brain and specific area of interest removed using asterile procedure. Brain areas of particular interest include any areafrom which progenitor cells can be obtained which will serve to restorefunction to a degenerated area of the host's brain. These regionsinclude areas of the central nervous system (CNS) including the cerebralcortex, cerebellum, midbrain, brainstem, spinal cord and ventriculartissue, and areas of the peripheral nervous system (PNS) including thecarotid body and the adrenal medulla. More particularly, these areasinclude regions in the basal ganglia, preferably the striatum whichconsists of the caudate and putamen, or various cell groups such as theglobus pallidus, the subthalamic nucleus, the nucleus basalis which isfound to be degenerated in Alzheimer's Disease patients, or thesubstantia nigra pars compacta which is found to be degenerated inParkinson's Disease patients.

Human heterologous neural progenitor cells may be derived from fetaltissue obtained from elective abortion, or from a post-natal, juvenileor organ donor. Autologous neural tissue can be obtained by biopsy, orfrom patients undergoing neurosurgery in which neural tissue is removed,in particular during epilepsy surgery, and more particularly duringtemporal lobectomies and hippocampalectomies.

Cells can be obtained from donor tissue by dissociation of individualcells from the connecting extracellular matrix of the tissue.Dissociation can be obtained using any known procedure, includingtreatment with enzymes such as trypsin, collagenase and the like, or byusing physical methods of dissociation such as with a blunt instrumentor by mincing with a scalpel to a allow outgrowth of specific cell typesfrom a tissue. Dissociation of fetal cells can be carried out in tissueculture medium, while a preferable medium for dissociation of juvenileand adult cells is artificial cerebral spinal fluid (aCSF). Regular aCSFcontains 124 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃,and 10 mM D-glucose. Low Ca²⁺ aCSF contains the same ingredients exceptfor Mg Cl₂ at a concentration of 3.2 mM and CaCl₂ at a concentration of0.1 mM.

Dissociated cells can be placed into any known culture medium capable ofsupporting cell growth, including MEM, DMEM, RPMI, F-12, and the like,containing supplements which are required for cellular metabolism suchas glutamine and other amino acids, vitamins, minerals and usefulproteins such as transferrin and the like. Medium may also containantibiotics to prevent contamination with yeast, bacteria and fungi suchas penicillin, streptomycin, gentamicin and the like. In some cases, themedium may contain serum derived from bovine, equine, chicken and thelike. A particularly preferable medium for cells is a mixture of DMEMand F-12.

Conditions for culturing should be close to physiological conditions.The pH of the culture media should be close to physiological pH,preferably between pH 6-8, more preferably close to pH 7, even moreparticularly about pH 7.4. Cells should be cultured at a temperatureclose to physiological temperature, preferably between 30° C.-40° C.,more preferably between 32° C.-38° C., and most preferably between 35°C.-37° C.

Cells can be grown in suspension or on a fixed substrate, butproliferation of the progenitors is preferably done in suspension togenerate large numbers of cells by formation of “neurospheres” (see, forexample, Reynolds et al. (1992) Science 255:1070-1709; and PCTPublications WO93/01275, WO94/09119, WO94/10292, and WO94/16718). In thecase of propagating (or splitting) suspension cells, flasks are shakenwell and the neurospheres allowed to settle on the bottom corner of theflask. The spheres are then transferred to a 50 ml centrifuge tube andcentrifuged at low speed. The medium is aspirated, the cells resuspendedin a small amount of medium with growth factor, and the cellsmechanically dissociated and resuspended in separate aliquots of media.

Cell suspensions in culture medium are supplemented with any growthfactor which allows for the proliferation of progenitor cells and seededin any receptacle capable of sustaining cells, though as set out above,preferably in culture flasks or roller bottles. Cells typicallyproliferate within 34 days in a 37° C. incubator, and proliferation canbe reinitiated at any time after that by dissociation of the cells andresuspension in fresh medium containing growth factors.

In the absence of substrate, cells lift off the floor of the flask andcontinue to proliferate in suspension forming a hollow sphere ofundifferentiated cells. After approximately 3-10 days in vitro, theproliferating clusters (neurospheres) are fed every 2-7 days, and moreparticularly every 2-4 days by gentle centrifugation and resuspension inmedium containing growth factor.

After 6-7 days in vitro, individual cells in the neurospheres can beseparated by physical dissociation of the neurospheres with a bluntinstrument, more particularly by triturating the neurospheres with apipette. Single cells from the dissociated neurospheres are suspended inculture medium containing growth factors, and differentiation of thecells can be control in culture by plating (or resuspending) the cellsin the presence of a subject compound.

To further illustrate other uses of the subject compounds, it is notedthat intracerebral grafting has emerged as an additional approach tocentral nervous system therapies. For example, one approach to repairingdamaged brain tissues involves the transplantation of cells from fetalor neonatal animals into the adult brain (Dunnett et al. (1987) J ExpBiol 123:265-289; and Freund et al. (1985) J Neurosci 5:603-616). Fetalneurons from a variety of brain regions can be successfully incorporatedinto the adult brain, and such grafts can alleviate behavioral defects.For example, movement disorder induced by lesions of dopaminergicprojections to the basal ganglia can be prevented by grafts of embryonicdopaminergic neurons. Complex cognitive functions that are impairedafter lesions of the neocortex can also be partially restored by graftsof embryonic cortical cells. The subject method can be used to regulatethe growth state in the culture, or where fetal tissue is used,especially neuronal stem cells, can be used to regulate the rate ofdifferentiation of the stem cells.

Stem cells useful in the present invention are generally known. Forexample, several neural crest cells have been identified, some of whichare multipotent and likely represent uncommitted neural crest cells, andothers of which can generate only one type of cell, such as sensoryneurons, and likely represent committed progenitor cells. The role ofcompounds employed in the present method to culture such stem cells canbe to regulate differentiation of the uncommitted progenitor, or toregulate further restriction of the developmental fate of a committedprogenitor cell towards becoming a terminally differentiated neuronalcell. For example, the present method can be used in vitro to regulatethe differentiation of neural crest cells into Glial cells, Schwanncells, Chromaffin cells, Cholinergic sympathetic or parasympatheticneurons, as well as peptidergic and serotonergic neurons. The subjectcompounds can be used alone, or can be used in combination with otherneurotrophic factors which act to more particularly enhance a particulardifferentiation fate of the neuronal progenitor cell.

In addition to the implantation of cells cultured in the presence of thesubject compounds, yet another aspect of the present invention concernsthe therapeutic application of a subject compound to regulate the growthstate of neurons and other neuronal cells in both the central nervoussystem and the peripheral nervous system. The ability of ptc, hedgehog,and smoothened to regulate neuronal differentiation during developmentof the nervous system and also presumably in the adult state indicatesthat, in certain instances, the subject compounds can be expected tofacilitate control of adult neurons with regard to maintenance,functional performance, and aging of normal cells; repair andregeneration processes in chemically or mechanically lesioned cells; andtreatment of degeneration in certain pathological conditions. In lightof this understanding, the present invention specifically contemplatesapplications of the subject method to the treatment protocol of(prevention and/or reduction of the severity of) neurological conditionsderiving from: (i) acute, subacute, or chronic injury to the nervoussystem, including traumatic injury, chemical injury, vascular injury anddeficits (such as the ischemia resulting from stroke), together withinfectious/inflammatory and tumor-induced injury, (ii) aging of thenervous system including Alzheimer's disease; (iii) chronicneurodegenerative diseases of the nervous system, including Parkinson'sdisease, Huntington's chorea, amylotrophic lateral sclerosis and thelike, as well as spinocerebellar degenerations; and (iv) chronicimmunological diseases of the nervous system or affecting the nervoussystem, including multiple sclerosis.

As appropriate, the subject method can also be used in generating nerveprostheses for the repair of central and peripheral nerve damage. Inparticular, where a crushed or severed axon is intubulated by use of aprosthetic device, subject compounds can be added to the prostheticdevice to regulate the rate of growth and regeneration of the dendriticprocesses. Exemplary nerve guidance channels are described in U.S. Pat.Nos. 5,092,871 and 4,955,892, incorporated herein by reference.

In another embodiment, the subject method can be used in the treatmentof neoplastic or hyperplastic transformations such as may occur in thecentral nervous system. For instance, the subject compounds can beutilized to cause such transformed cells to become either post-mitoticor apoptotic. The present method may, therefore, be used as part of atreatment for, e.g., malignant gliomas, meningiomas, medulloblastomas,neuroectodermal tumors, and ependymomas, etc. In this connection, theinvention still further relates to inducing antiangiogenesis inlocalized or distant metastasized tumors by affecting cancer relatedvascular cells.

In a preferred embodiment, the subject method can be used as part of atreatment regimen for malignant medulloblastoma and other primary CNSmalignant neuroectodermal tumors.

In certain embodiments, the subject method is used as part of treatmentprogram for medulloblastoma. Medulloblastoma, a primary brain tumor, isthe most common brain tumor in children. A medulloblastoma is aprimitive neuroectodermal tumor arising in the posterior fossa. Theyaccount for approximately 25% of all pediatric brain tumors (Miller).Histologically, they are small round cell tumors commonly arranged intrue rosettes, but may display some differentiation to astrocytes,ependymal cells or neurons (Rorke; Kleihues). PNETs may arise in otherareas of the brain including the pineal gland (pineoblastoma) andcerebrum. Those arising in the supratentorial region generally fareworse than their PF counterparts.

Medulloblastoma/PNETs are known to recur anywhere in the CNS alterresection, and can even metastasize to bone. Pretreatment evaluationshould therefore include an examination of the spinal cord to excludethe possibility of “dropped metastases”. Gadolinium-enhanced MRI haslargely replaced myelography for this purpose, and CSF cytology isobtained postoperatively as a routine procedure.

In other embodiments, the subject method is used as part of treatmentprogram for ependymomas. Ependymomas account for approximately 10% ofthe pediatric brain tumors in children. Grossly, they are tumors thatarise from the ependymal lining of the ventricles and microscopicallyform rosettes, canals, and perivascular rosettes. Of the CHOP series of51 children reported with ependymomas, not all are malignant andapproximately ⅔ arise from the region of the 4th ventricle. One thirdpresented in the supratentorial region. Age at presentation peaksbetween birth and 4 years, as demonstrated by SEER data as well as datafrom CHOP. The median age is about 5 years. Because so many childrenwith this disease are babies, they often require multimodal therapy.

Yet another aspect of the present invention concerns the observation inthe art that ptc, hedgehog, and/or smoothened are involved inmorphogenic signals involved in other vertebrate organogenic pathways inaddition to neuronal differentiation as described above, having apparentroles in other endodermal patterning, as well as both mesodermal andendodermal differentiation processes. Thus, it is contemplated by theinvention that compositions comprising one or more of the subjectcompounds can also be utilized for both cell culture and therapeuticmethods involving generation and maintenance of non-neuronal tissue.

In one embodiment, the present invention makes use of the discovery thatptc, hedgehog, and smoothened are apparently involved in controlling thedevelopment of stem cells responsible for formation of the digestivetract, liver, lungs, and other organs which derive from the primitivegut. Shh serves as an inductive signal from the endoderm to themesoderm, which is critical to gut morphogenesis. Therefore, forexample, compounds of the instant method can be employed for regulatingthe development and maintenance of an artificial liver which can havemultiple metabolic functions of a normal liver. In an exemplaryembodiment, the subject method can be used to regulate the proliferationand differentiation of digestive tube stem cells to form hepatocytecultures which can be used to populate extracellular matrices, or whichcan be encapsulated in biocompatible polymers, to form both implantableand extracorporeal artificial livers.

In another embodiment, therapeutic compositions of subject compounds canbe utilized in conjunction with transplantation of such artificiallivers, as well as embryonic liver structures, to regulate uptake ofintraperitoneal implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted liver tissue.

In yet another embodiment, the subject method can be employedtherapeutically to regulate such organs after physical, chemical orpathological insult. For instance, therapeutic compositions comprisingsubject compounds can be utilized in liver repair subsequent to apartial hepatectomy.

The generation of the pancreas and small intestine from the embryonicgut depends on intercellular signalling between the endodermal andmesodermal cells of the gut. In particular, the differentiation ofintestinal mesoderm into smooth muscle has been suggested to depend onsignals from adjacent endodermal cells. One candidate mediator ofendodermally derived signals in the embryonic hindgut is Sonic hedgehog.See, for example, Apelqvist et al. (1997) Curr Biol 7:801-4. The Shhgene is expressed throughout the embryonic gut endoderm with theexception of the pancreatic bud endoderm, which instead expresses highlevels of the homeodomain protein Ipf1/Pdx1 (insulin promoter factor1/pancreatic and duodenal homeobox 1), an essential regulator of earlypancreatic development. Apelqvist et al., supra, have examined whetherthe differential expression of Shh in the embryonic gut tube controlsthe differentiation of the surrounding mesoderm into specialisedmesoderm derivatives of the small intestine and pancreas. To test this,they used the promoter of the Ipf1/Pdx1 gene to selectively express Shhin the developing pancreatic epithelium. In Ipf1/Pdx1-Shh transgenicmice, the pancreatic mesoderm developed into smooth muscle andinterstitial cells of Caja1, characteristic of the intestine, ratherthan into pancreatic mesenchyme and spleen. Also, pancreatic explantsexposed to Shh underwent a similar program of intestinaldifferentiation. These results provide evidence that the differentialexpression of endodermally derived Shh controls the fate of adjacentmesoderm at different regions of the gut tube.

In the context of the present invention, it is contemplated thereforethat the subject compounds can be used to control or regulate theproliferation and/or differentiation of pancreatic tissue and intestinaltissue (see distal hindgut deformation discussed elsewhere within thepresent specification) both in vivo and in vitro.

There are a wide variety of pathological cell proliferative anddifferentiative conditions for which the inhibitors of the presentinvention may provide therapeutic benefits, with the general strategybeing, for example, the correction of aberrant insulin expression, ormodulation of differentiation. More generally, however, the presentinvention relates to a method of inducing and/or maintaining adifferentiated state, enhancing survival and/or affecting proliferationof pancreatic cells, by contacting the cells with the subjectinhibitors. For instance, it is contemplated by the invention that, inlight of the apparent involvement of ptc, hedgehog, and smoothened inthe formation of ordered spatial arrangements of pancreatic tissues, thesubject method is suitable for use as part of a technique to generateand/or maintain such tissue both in vitro and in vivo. For instance,modulation of the function of hedgehog can be employed in both cellculture and therapeutic methods involving generation and maintenanceβ-cells and possibly also for non-pancreatic tissue, such as incontrolling the development and maintenance of tissue from the digestivetract, spleen, lungs, urogenital organs (e.g., bladder), and otherorgans which derive from the primitive gut.

In an exemplary embodiment, the present method can be used in thetreatment of hyperplastic and neoplastic disorders effecting pancreatictissue, particularly those characterized by aberrant proliferation ofpancreatic cells. For instance, pancreatic cancers are marked byabnormal proliferation of pancreatic cells which can result inalterations of insulin secretory capacity of the pancreas. For instance,certain pancreatic hyperplasias, such as pancreatic carcinomas, canresult in hypoinsulinemia due to dysfunction of .beta.-cells ordecreased islet cell mass. To the extent that one more of aberrantPI3K/hedgehog, ptc, smoothened signaling may be indicated in diseaseprogression, the subject regulators can be used to enhance regenerationof the tissue after anti-tumor therapy.

Moreover, manipulation of PI3K/hedgehog signaling properties atdifferent points may be useful as part of a strategy forreshaping/repairing pancreatic tissue both in vivo and in vitro. In oneembodiment, the present invention makes use of the apparent involvementof ptc, hedgehog, and smoothened in regulating the development ofpancreatic tissue. In general, the subject method can be employedtherapeutically to regulate the pancreas after physical, chemical orpathological insult. In yet another embodiment, the subject method canbe applied to cell culture techniques, and in particular, may beemployed to enhance the initial generation of prosthetic pancreatictissue devices. Manipulation of proliferation and differentiation ofpancreatic tissue, for example, by altering hedgehog activity, canprovide a means for more carefully controlling the characteristics of acultured tissue. In an exemplary embodiment, the subject method can beused to augment production of prosthetic devices which require β-isletcells, such as may be used in the encapsulation devices described in,for example, Aebischer et al. U.S. Pat. No. 4,892,538, Aebischer et al.U.S. Pat. No. 5,106,627; Lim U.S. Pat. No. 4,391,909, the Sefton U.S.Pat. No. 4,353,888, all incorporated herein by reference. Earlyprogenitor cells to the pancreatic islets are multipotential, andapparently coactivate all the islet-specific genes from the time theyfirst appear. As development proceeds, expression of islet-specifichormones, such as insulin, becomes restricted to the pattern ofexpression characteristic of mature islet cells. The phenotype of matureislet cells, however, is not stable in culture, as reappearance ofembryonal traits in mature β-cells can be observed. By utilizing thesubject compounds, the differentiation path or proliferative index ofthe cells can be regulated.

Furthermore, manipulation of the differentiative state of pancreatictissue can be utilized in conjunction with transplantation of artificialpancreas so as to promote implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted tissue. For instance,manipulation of hedgehog function to affect tissue differentiation canbe utilized as a means of maintaining graft viability.

Bellusci et al. (1997) Development 124:53 report that Sonic hedgehogregulates lung mesenchymal cell proliferation in vivo. Accordingly, thepresent method can be used to regulate regeneration of lung tissue,e.g., in the treatment of emphysema.

Fujita et al. (1997) Biochem Biophys Res Commun 238:658 reported thatSonic hedgehog is expressed in human lung squamous carcinoma andadenocarcinoma cells. The expression of Sonic hedgehog was also detectedin the human lung squamous carcinoma tissues, but not in the normal lungtissue of the same patient. They also observed that Sonic hedgehogstimulates the incorporation of BrdU into the carcinoma cells andstimulates their cell growth, while anti-Shh-N inhibited their cellgrowth. These results suggest that a ptc, hedgehog, and/or smoothened isinvolved in the cell growth of such transformed lung tissue andtherefore indicates that the subject method can be used as part of atreatment of lung carcinoma and adenocarcinomas, and other proliferativedisorders involving the lung epithelia.

Numerous other tumors may (based on evidence such as involvement of thehedgehog pathway in these tumors, or detected expression of hedgehog orits receptor in these tissues during development) be affected bytreatment with the subject compounds. Such tumors include, but are by nomeans limited to, tumors related to Gorlin's syndrome (e.g., basal cellcarcinoma, medulloblastoma, meningioma, etc.), tumors evidenced in pctknock-out mice (e.g., hemangioma, rhabdomyosarcoma, etc.), tumorsresulting from gli-I amplification (e.g., glioblastoma, sarcoma, etc.),tumors connected with TRC8, a ptc homolog (e.g., renal carcinoma,thyroid carcinoma, etc.), Ext-1-related tumors (e.g., bone cancer,etc.), Shh-induced tumors (e.g., lung cancer, chondrosarcomas, etc.),and other tumors (e.g., breast cancer, urogenital cancer (e.g., kidney,bladder, ureter, prostate, etc.), adrenal cancer, gastrointestinalcancer (e.g., stomach, intestine, etc.), etc.).

In still another embodiment of the present invention, compositionscomprising one or more of the subject compounds can be used in the invitro generation of skeletal tissue, such as from skeletogenic stemcells, as well as the in vivo treatment of skeletal tissue deficiencies.The present invention particularly contemplates the use of subjectcompounds to regulate the rate of chondrogenesis and/or osteogenesis. By“skeletal tissue deficiency”, it is meant a deficiency in bone or otherskeletal connective tissue at any site where it is desired to restorethe bone or connective tissue, no matter how the deficiency originated,e.g. whether as a result of surgical intervention, removal of tumor,ulceration, implant, fracture, or other traumatic or degenerativeconditions.

For example, the method of the present invention can be used as part ofa regimen for restoring cartilage function to a connective tissue. Suchmethods are useful in, for example, the repair of defects or lesions incartilage tissue which is the result of degenerative wear such as thatwhich results in arthritis, as well as other mechanical derangementswhich may be caused by trauma to the tissue, such as a displacement oftorn meniscus tissue, meniscectomy, a Taxation of a joint by a tornligament, malignment of joints, bone fracture, or by hereditary disease.The present reparative method is also useful for remodeling cartilagematrix, such as in plastic or reconstructive surgery, as well asperiodontal surgery. The present method may also be applied to improvinga previous reparative procedure, for example, following surgical repairof a meniscus, ligament, or cartilage. Furthermore, it may prevent theonset or exacerbation of degenerative disease if applied early enoughafter trauma.

In one embodiment of the present invention, the subject method comprisestreating the afflicted connective tissue with a therapeuticallysufficient amount of a subject compound to regulate a cartilage repairresponse in the connective tissue by managing the rate ofdifferentiation and/or proliferation of chondrocytes embedded in thetissue. Such connective tissues as articular cartilage, interarticularcartilage (menisci), costal cartilage (connecting the true ribs and thesternum), ligaments, and tendons are particularly amenable to treatmentin reconstructive and/or regenerative therapies using the subjectmethod. As used herein, regenerative therapies include treatment ofdegenerative states which have progressed to the point of whichimpairment of the tissue is obviously manifest, as well as preventivetreatments of tissue where degeneration is in its earliest stages orimminent.

In an illustrative embodiment, the subject method can be used as part ofa therapeutic intervention in the treatment of cartilage of adiarthroidal joint, such as a knee, an ankle, an elbow, a hip, a wrist,a knuckle of either a finger or toe, or a tempomandibular joint. Thetreatment can be directed to the meniscus of the joint, to the articularcartilage of the joint, or both. To further illustrate, the subjectmethod can be used to treat a degenerative disorder of a knee, such aswhich might be the result of traumatic injury (e.g., a sports injury orexcessive wear) or osteoarthritis. The subject regulators may beadministered as an injection into the joint with, for instance, anarthroscopic needle. In some instances, the injected agent can be in theform of a hydrogel or other slow release vehicle described above inorder to permit a more extended and regular contact of the agent withthe treated tissue.

The present invention further contemplates the use of the subject methodin the field of cartilage transplantation and prosthetic devicetherapies. However, problems arise, for instance, because thecharacteristics of cartilage and fibrocartilage varies between differenttissue: such as between articular, meniscal cartilage, ligaments, andtendons, between the two ends of the same ligament or tendon, andbetween the superficial and deep parts of the tissue. The zonalarrangement of these tissues may reflect a gradual change in mechanicalproperties, and failure occurs when implanted tissue, which has notdifferentiated under those conditions, lacks the ability toappropriately respond. For instance, when meniscal cartilage is used torepair anterior cruciate ligaments, the tissue undergoes a metaplasia topure fibrous tissue. By regulating the rate of chondrogenesis, thesubject method can be used to particularly address this problem, byhelping to adaptively control the implanted cells in the new environmentand effectively resemble hypertrophic chondrocytes of an earlierdevelopmental stage of the tissue.

In similar fashion, the subject method can be applied to enhancing boththe generation of prosthetic cartilage devices and to theirimplantation. The need for improved treatment has motivated researchaimed at creating new cartilage that is based oncollagen-glycosaminoglycan templates (Stone et al. (1990) Clin OrthopRelat Red 252:129), isolated chondrocytes (Grande et al. (1989) J OrthopRes 7:208; and Takigawa et al. (1987) Bone Miner 2:449), andchondrocytes attached to natural or synthetic polymers (Walitani et al.(1989) J Bone Jt Surg 71B:74; Vacanti et al. (1991) Plast Reconstr Surg88:753; von Schroeder et al. (1991) J Biomed Mater Res 25:329; Freed etal. (1993) 3 Biomed Mater Res 27:11; and the Vacanti et al. U.S. Pat.No. 5,041,138). For example, chondrocytes can be grown in culture onbiodegradable, biocompatible highly porous scaffolds formed frompolymers such as polyglycolic acid, polylactic acid, agarose gel, orother polymers which degrade over time as function of hydrolysis of thepolymer backbone into innocuous monomers. The matrices are designed toallow adequate nutrient and gas exchange to the cells until engraftmentoccurs. The cells can be cultured in vitro until adequate cell volumeand density has developed for the cells to be implanted. One advantageof the matrices is that they can be cast or molded into a desired shapeon an individual basis, so that the final product closely resembles thepatient's own ear or nose (by way of example), or flexible matrices canbe used which allow for manipulation at the time of implantation, as ina joint.

In one embodiment of the subject method, the implants are contacted witha subject compound during certain stages of the culturing process inorder to manage the rate of differentiation of chondrocytes and theformation of hypertrophic chrondrocytes in the culture.

In another embodiment, the implanted device is treated with a subjectcompound in order to actively remodel the implanted matrix and to makeit more suitable for its intended function. As set out above withrespect to tissue transplants, the artificial transplants suffer fromthe same deficiency of not being derived in a setting which iscomparable to the actual mechanical environment in which the matrix isimplanted. The ability to regulate the chondrocytes in the matrix by thesubject method can allow the implant to acquire characteristics similarto the tissue for which it is intended to replace.

In yet another embodiment, the subject method is used to enhanceattachment of prosthetic devices. To illustrate, the subject method canbe used in the implantation of a periodontal prosthesis, wherein thetreatment of the surrounding connective tissue stimulates formation ofperiodontal ligament about the prosthesis.

In still further embodiments, the subject method can be employed as partof a regimen for the generation of bone (osteogenesis) at a site in theanimal where such skeletal tissue is deficient Indian hedgehog isparticularly associated with the hypertrophic chondrocytes that areultimately replaced by osteoblasts. For instance, administration of acompound of the present invention can be employed as part of a methodfor regulating the rate of bone loss in a subject. For example,preparations comprising subject compounds can be employed, for example,to control endochondral ossification in the formation of a “model” forossification.

In yet another embodiment of the present invention, a subject compoundcan be used to regulate spermatogenesis. The hedgehog proteins,particularly Dhh, have been shown to be involved in the differentiationand/or proliferation and maintenance of testicular germ cells. Dhhexpression is initiated in Sertoli cell precursors shortly after theactivation of Sry (testicular determining gene) and persists in thetestis into the adult. Azospermic and oligospermic males are viable butinfertile, owing to a complete absence of mature sperm. Examination ofthe developing testis in different genetic backgrounds suggests that Dhhregulates both early and late stages of spermatogenesis. Bitgood et al.(1996) Curr Biol 6:298. In a preferred embodiment, the subject compoundcan be used as a contraceptive. In a similar fashion, compounds of thesubject method are potentially useful for modulating abnormal ovarianfunction, and at the same time, offering protective effects against theuse of ovulation inductors for treating infertility in phenotypes withreceptor loss of function (non-limiting drug list incorporated herein).

In yet another embodiment of the present invention, a subject compoundcan be used to regulate ovulation as describe in the preceedingparagraph. The hedgehog proteins, particularly Dhh, have been shown tobe involved induced expression of the hedgehog target genes Ptch1 andGli1, in the surrounding pre-theca cell compartment. Cyclopamine, ahighly specific hedgehog signaling antagonist, inhibits this inducedexpression of target genes in cultured neonatal mouse ovaries. The thecacell compartment remains a target of hedgehog signaling throughoutfollicle development, showing induced expression of the hedgehog targetgenes Ptch1, Ptch2, Hip1, and Gli1. In periovulatory follicles, adynamic synchrony between loss of hedgehog expression and loss ofinduced target gene expression is observed. Oocytes are unable torespond to hedgehog because they lack expression of the essential signaltransducer Smo (smoothened). The present results point to a prominentrole of hedgehog signaling in the communication between granulosa cellsand developing theca cells (Endocrinology Vol. 146, No. 8 3558-3566,2005).

The subject method also has wide applicability to the treatment orprophylaxis of disorders afflicting epithelial tissue, as well as incosmetic uses. In general, the method can be characterized as includinga step of administering to an animal an amount of a subject compoundeffective to alter the growth state of a treated epithelial tissue. Themode of administration and dosage regimens will vary depending on theepithelial tissue(s) which is to be treated. For example, topicalformulations will be preferred where the treated tissue is epidermaltissue, such as dermal or mucosal tissues.

A method which “promotes the healing of a wound” results in the woundhealing more quickly as a result of the treatment than a similar woundheals in the absence of the treatment. “Promotion of wound healing” canalso mean that the method regulates the proliferation and/or growth of,inter alia, keratinocytes, or that the wound heals with less scarring,less wound contraction, less collagen deposition and more superficialsurface area. In certain instances, “promotion of wound healing” canalso mean that certain methods of wound healing have improved successrates, (e.g., the take rates of skin grafts) when used together with themethod of the present invention. (See the earlier discussion concerningfibroblasts and wound healing above.)

Despite significant progress in reconstructive surgical techniques,scarring can be an important obstacle in regaining normal function andappearance of healed skin. This is particularly true when pathologicscarring such as keloids or hypertrophic scars of the hands or facecauses functional disability or physical deformity. In the severestcircumstances, such scarring may precipitate psychosocial distress and alife of economic deprivation. Wound repair includes the stages ofhemostasis, inflammation, proliferation, and remodeling. Theproliferative stage involves multiplication of fibroblasts andendothelial and epithelial cells. Through the use of the subject method,the rate of proliferation of epithelial cells in and proximal to thewound can be controlled in order to accelerate closure of the woundand/or minimize the formation of scar tissue.

The subject method can also be used in the treatment of corneopathicsmarked by corneal epithelial cell proliferation, as for example inocular epithelial disorders such as epithelial downgrowth or squamouscell carcinomas of the ocular surface. Also, for example, the subjectmethod and compounds can be used to treat degenerative diseases of theretina.

Levine et al. (1997) J Neurosci 17:6277 show that hedgehog proteins canregulate mitogenesis and photoreceptor differentiation in the vertebrateretina, and Ihh is a candidate factor from the pigmented epithelium topromote retinal progenitor proliferation and photoreceptordifferentiation. Likewise, Jensen et al. (1997) Development 124:363demonstrated that treatment of cultures of perinatal mouse retinal cellswith the amino-terminal fragment of Sonic hedgehog results in anincrease in the proportion of cells that incorporate bromodeoxuridine,in total cell numbers, and in rod photoreceptors, amacrine cells andMuller glia1 cells, suggesting that Sonic hedgehog promotes theproliferation of retinal precursor cells. Thus, the subject method canbe used in the treatment of proliferative diseases of retinal cells andregulate photoreceptor differentiation. Furthermore, Bennett, Jeffrey L.Journal of Neuro-Ophthalmology: Volume 22(4) December 2002 pp 286-296cites recent studies that have identified several factors important forthe determination and function of the optic disc: sonic hedgehog (Shh),Pax Gli3 transcription factors. Deficient Shh expression in zebrafishleads to cyclopia, whereas ectopic expression results in small opticcups and enlarged optic stalks. Pax2 null mutant mice fail to form opticdiscs, resulting in medial extension of retinal pigment epithelial cellsinto the optic stalk, failure of axons to cross at the optic chiasm, andoptic nerve coloboma. The murine Gli3 mutant, ‘extra-toes,’ also hasoptic nerve coloboma). Mutations in the human Shh and Pax2 genes areknown to result in holoprosencephaly and the renal-coloboma syndrome,and possibly retinoblastoma. Retinitis Pigmentosa is a photoreceptordegenerative disease leading to blindness in adulthood. Ala Moshiri etal, The Journal of Neuroscience, Jan. 7, 2004, 24(1):229-237;doi:10.1523/JNEUROSCI.2980-03.2004 also postulates that the hedgehogsignaling pathway is a key regulator of neural development, affectingboth proliferation and differentiation of neural progenitors. Sonichedgehog (Shh) is a mitogenic factor for retinal progenitors in vitro.They wanted to determine whether this signaling system is important invivo for regulating retinal progenitor proliferation, they analyzed micewith a single functional allele of the Shh receptor patched (ptc). Theyfound that ptc+/− mice had increased numbers of neural progenitors atevery stage of retinal development that they examined. In addition,these mice had persistent progenitors at the retinal margin for up to 3months of age, reminiscent of the ciliary marginal zone of lowervertebrates. To test whether the progenitors at the retinal margin ofptc+/− mice could be induced to regenerate retinal neurons in responseto damage, they bred ptc+/− mice onto a retinal degeneration background(pro23his rhodopsin transgenic) and labeled newly generated cells withcombined immunohistochemistry for bromodeoxyuridine and retinal neuronand photoreceptor-specific markers. Ala Moshiri et al, (2004) foundnewly generated neurons and photoreceptors at the retinal margin inptc+/−; pro23his mice. They propose that the Shh pathway may act as aregulator of both prenatal and postnatal retinal growth. Through the useof the subject method and compounds, there is hope in treating diseasesassociated with degeneration of the photoreceptors and for treatingother childhood cancers like retinoblastoma related to deficiencies inShh pathways.

The subject method and compositions can also be used to treat woundsresulting from dermatological diseases, such as lesions resulting fromautoimmune disorders such as psoriasis. Atopic dermititis refers to skintrauma resulting from allergies associated with an immune responsecaused by allergens such as pollens, foods, dander, insect venoms andplant toxins.

In another aspect of the invention, the subject method can be used toinduce differentiation and/or inhibit proliferation of epitheliallyderived tissue. Such forms of these molecules can provide a basis fordifferentiation therapy for the treatment of hyperplastic and/orneoplastic conditions involving epithelial tissue. For example, suchpreparations can be used for the treatment of cutaneous diseases inwhich there is abnormal proliferation or growth of cells of the skin.

Yet another aspect of the present invention relates to the use of thesubject method to control hair growth. Hair is basically composed ofkeratin, a tough and insoluble protein; its chief strength lies in itsdisulphide bond of cystine. Each individual hair comprises a cylindricalshaft and a root, and is contained in a follicle, a flask-likedepression in the skin. The bottom of the follicle contains afinger-like projection termed the papilla, which consists of connectivetissue from which hair grows, and through which blood vessels supply thecells with nourishment The shaft is the part that extends outwards fromthe skin surface, whilst the root has been described as the buried partof the hair. The base of the root expands into the hair bulb, whichrests upon the papilla. Cells from which the hair is produced grow inthe bulb of the follicle; they are extruded in the form of fibers as thecells proliferate in the follicle. Hair “growth” refers to the formationand elongation of the hair fiber by the dividing cells.

As is well known in the art, the common hair cycle is divided into threestages: anagen, catagen and telogen. During the active phase (anagen),the epidermal stem cells of the dermal papilla divide rapidly. Daughtercells move upward and differentiate to form the concentric layers of thehair itself. The transitional stage, catagen, is marked by the cessationof mitosis of the stem cells in the follicle. The resting stage is knownas telogen, where the hair is retained within the scalp for severalweeks before an emerging new hair developing below it dislodges thetelogen-phase shaft from its follicle. From this model it has becomeclear that the larger the pool of dividing stem cells that differentiateinto hair cells, the more hair growth occurs. Accordingly, methods forincreasing or reducing hair growth can be carried out by potentiating orinhibiting, respectively, the proliferation of these stem cells.

In certain embodiments, the subject method can be employed as a way ofreducing the growth of human hair as opposed to its conventional removalby cutting, shaving, or depilation. For instance, the present method canbe used in the treatment of trichosis characterized by abnormally rapidor dense growth of hair, e.g. hypertrichosis. In an exemplaryembodiment, subject compounds can be used to manage hirsutism, adisorder marked by abnormal hairiness. The subject method can alsoprovide a process for extending the duration of depilation.

Moreover, because a subject compound will often be cytostatic toepithelial cells, rather than cytotoxic, such agents can be used toprotect hair follicle cells from cytotoxic agents which requireprogression into S-phase of the cell-cycle for efficacy, e.g.radiation-induced death. Treatment by the subject method can provideprotection by causing the hair follicle cells to become quiescent, e.g.,by inhibiting the cells from entering S phase, and thereby preventingthe follicle cells from undergoing mitotic catastrophe or programmedcell death. For instance, subject compounds can be used for patientsundergoing chemo- or radiation-therapies which ordinarily result in hairloss. By inhibiting cell-cycle progression during such therapies, thesubject treatment can protect hair follicle cells from death which mightotherwise result from activation of cell death programs. After thetherapy has concluded, the instant method can also be removed withconcommitant relief of the inhibition of follicle cell proliferation.

The subject method can also be used in the treatment of folliculitis,such as folliculitis decalvans, folliculitis ulerythematosa reticulataor keloid folliculitis. For example, a cosmetic preparation of a subjectcompound can be applied topically in the treatment ofpseudofolliculitis, a chronic disorder occurring most often in thesubmandibular region of the neck and associated with shaving, thecharacteristic lesions of which are erythematous papules and pustulescontaining buried hairs.

In another aspect of the invention, the subject method can be used toinduce differentiation and/or inhibit proliferation of epitheliallyderived tissue. Such forms of these molecules can provide a basis fordifferentiation therapy for the treatment of hyperplastic and/orneoplastic conditions involving epithelial tissue. For example, suchpreparations can be used for the treatment of cutaneous diseases inwhich there is abnormal proliferation or growth of cells of the skin.

For instance, the pharmaceutical preparations of the invention areintended for the treatment of hyperplastic epidermal conditions, such askeratosis, as well as for the treatment of neoplastic epidermalconditions such as those characterized by a high proliferation rate forvarious skin cancers, as for example basal cell carcinoma or squamouscell carcinoma. The subject method can also be used in the treatment ofautoimmune diseases affecting the skin, in particular, of dermatologicaldiseases involving morbid proliferation and/or keratinization of theepidermis, as for example, caused by psoriasis or atopic dermatosis.

Many common diseases of the skin, such as psoriasis, squamous cellcarcinoma, keratoacanthoma and actinic keratosis are characterized bylocalized abnormal proliferation and growth. For example, in psoriasis,which is characterized by scaly, red, elevated plaques on the skin, thekeratinocytes are known to proliferate much more rapidly than normal andto differentiate less completely.

In one embodiment, the preparations of the present invention aresuitable for the treatment of dermatological ailments linked tokeratinization disorders causing abnormal proliferation of skin cells,which disorders may be marked by either inflammatory or non-inflammatorycomponents. To illustrate, therapeutic preparations of a subjectcompound, e.g., which promotes quiescense or differentiation, can beused to treat varying forms of psoriasis, be they cutaneous, mucosal orungual. Psoriasis, as described above, is typically characterized byepidermal keratinocytes which display marked proliferative activationand differentiation along a “regenerative” pathway. Treatment with anantiproliferative embodiment of the subject method can be used toreverse the pathological epidermal activation and can provide a basisfor sustained remission of the disease.

A variety of other keratotic lesions are also candidates for treatmentwith the subject method. Actinic keratoses, for example, are superficialinflammatory premalignant tumors arising on sun-exposed and irradiatedskin. The lesions are erythematous to brown with variable scaling.Current therapies include excisional and cryosurgery. These treatmentsare painful, however, and often produce cosmetically unacceptablescarring. Accordingly, treatment of keratosis, such as actinickeratosis, can include application, preferably topical, of a subjectcompound composition in amounts sufficient to inhibit hyperproliferationof epidermal/epidermoid cells of the lesion.

Acne represents yet another dermatologic ailment which may be treated bythe subject method. Acne vulgaris, for instance, is a multifactorialdisease most commonly occurring in teenagers and young adults, and ischaracterized by the appearance of inflammatory and noninflammatorylesions on the face and upper trunk. The basic defect which gives riseto acne vulgaris is hypercornification of the duct of a hyperactivesebaceous gland. Hypercornification blocks the normal mobility of skinand follicle microorganisms, and in so doing, stimulates the release oflipases by Propinobacterium acnes and Staphylococcus epidermidisbacteria and Pitrosporum ovale, a yeast. Treatment with anantiproliferative subject compound, particularly topical preparations,may be useful for preventing the transitional features of the ducts,e.g. hypercornification, which lead to lesion formation. The subjecttreatment may further include, for example, antibiotics, retinoids andantiandrogens.

The present invention also provides a method for treating various formsof dermatitis. Dermatitis is a descriptive term referring to poorlydemarcated lesions which are either pruritic, erythematous, scaly,blistered, weeping, fissured or crusted. These lesions arise from any ofa wide variety of causes. The most common types of dermatitis areatopic, contact and diaper dermatitis. For instance, seborrheicdermatitis is a chronic, usually pruritic, dermatitis with erythema,dry, moist, or greasy scaling, and yellow crusted patches on variousareas, especially the scalp, with exfoliation of an excessive amount ofdry scales. The subject method can also be used in the treatment ofstasis dermatitis, an often chronic, usually eczematous dermatitis.Actinic dermatitis is dermatitis that due to exposure to actinicradiation such as that from the sun, ultraviolet waves or x- orgamma-radiation. According to the present invention, the subject methodcan be used in the treatment and/or prevention of certain symptoms ofdermatitis caused by unwanted proliferation of epithelial cells. Suchtherapies for these various forms of dermatitis can also include topicaland systemic corticosteroids, antipurities, and antibiotics.

Ailments which may be treated by the subject method are disordersspecific to non-humans, such as mange as well as any of the diseasestates or conditions occurring in animals corresponding to theconditions and disease states in humans described above or below.

In still another embodiment, the subject method can be used in thetreatment of human cancers, particularly basal cell carcinomas and othertumors of epithelial tissues such as the skin. For example, subjectcompounds can be employed, in the subject method, as part, of atreatment for basal cell nevus syndrome (BCNS), and other humancarcinomas, adenocarcinomas, sarcomas and the like.

In a preferred embodiment, the subject method is used as part of atreatment at prophylaxis regimen for treating (or preventing) basal cellcarcinoma. The deregulation of the hedgehog signaling pathway may be ageneral feature of basal cell carcinomas caused by ptc mutations.Consistent overexpression of human ptc mRNA has been described in tumorsof familial and sporadic BCCs, determined by in situ hybridization.Mutations that inactivate ptc may be expected to result inoverexpression of mutant Ptc, because ptc displays negativeautoregulation. Prior research demonstrates that overexpression ofhedgehog proteins can also lead to tumorigenesis. That sonic hedgehog(Shh) has a role in tumorigenesis in the mouse has been suggested byresearch in which transgenic mice overexpressing Shh in the skindeveloped features of BCNS, including multiple BCC-like epidermalproliferations over the entire skin surface, after only a few days ofskin development. A mutation in the Shh human gene from a BCC was alsodescribed; it was suggested that Shh or other Hh genes in humans couldact as dominant oncogenes in humans. Sporadic ptc mutations have alsobeen observed in BCCs from otherwise normal individuals, some of whichare UV-signature mutations. In one recent study of sporadic BCCs, fiveUV-signature type mutations, either CT or CCTT changes, were found outof fifteen tumors determined to contain ptc mutations. Another recentanalysis of sporadic ptc mutations in BCCs and neuroectodermal tumorsrevealed one CT change in one of three ptc mutations found in the BCCs.See, for example, Goodrich et al. (1997) Science 277:1109-13; Xie et al.(1997) Cancer Res 57:2369-72; Oro et al. (1997) Science 276:817-21; Xieet al. (1997) Genes Chromosomes Cancer 18:305-9; Stone et al. (1996)Nature 384:129-34; and Johnson et al. Science 272:1668-71.

The subject method can also be used to treat patients with BCNS, e.g.,to prevent BCC or other effects of the disease which may be the resultof ptc loss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function. Basal cell nevus syndrome is a rare autosomal dominantdisorder characterized by multiple BCCs that appear at a young age. BCNSpatients are very susceptible to the development of these tumors; in thesecond decade of life, large numbers appear, mainly on sun-exposed areasof the skin. This disease also causes a number of developmentalabnormalities, including rib, head and face alterations, and sometimespolydactyly, syndactyly, and spina bifida. They also develop a number oftumor types in addition to BCCs: fibromas of the ovaries and heart,cysts of the skin and jaws, and in the central nervous system,medulloblastomas and meningiomas. The subject method can be used toprevent or treat such tumor types in BCNS and non-BCNS patients. Studiesof BCNS patients show that they have both genomic and sporadic,mutations in the ptc gene, suggesting that these mutations are theultimate cause of this disease.

In another aspect, the present invention provides pharmaceuticalpreparations and methods for controlling the formation ofmegakaryocyte-derived cells and/or controlling the functionalperformance of megakaryocyte-derived cells. For instance, certain of thecompositions disclosed herein may be applied to the treatment orprevention of a variety hyperplastic or neoplastic conditions affectingplatelets.

In certain embodiments, the invention compound can be chosen on thebasis of selectivity for the hedgehog pathway. This selectivity can befor the hedgehog pathway vs other mediated pathways that are used withthe compound as well as selectivity for particular hedgehog pathways,e.g., which isotype specific for hedgehog (e.g., Shh, Ihh, Dhh) or thepatched receptor (e.g., ptc-1, ptc-2). For instance, the subject methodmay employ different compounds with different phosphates which do notinterfere with the biological activity of compounds used in birthcontrol, chemotherapeutic agents, or other ablative therapies (withoutlimitation incorporated herein): Aldosterone, androstane, androstene,androstenedione, androsterone, cholecalciferol, cholestane, cholic acid,corticosterone, cortisol, cortisol acetate, cortisone, cortisoneacetate, deoxycorticosterone, digitoxigenin, ergocalciferol, ergosterol,estradiol-17-α, estradiol-17-β, estriol, estrane, estrone,hydrocortisone, lanosterol, lithocholic acid, mestranol, β-methasone,prednisone, pregnane, pregnenolone, progesterone, spironolactone (asused in Lybrel®), testosterone, triamcinolone and their derivatives.

In this manner, untoward side effects which may be associated withcertain members of these steroidal alkaloids, cancer therapeutics, andother class of drugs described above and incorporated herein can bereduced by using the D-chiroinositol compound. For example, some methodsand compostions may be employed as a means of reducing such unwantednegative side effects to certain drug regimes during chemotherapy orinfertility treatments. These side effects include hirsuitism (excesshair growth due to hormones), shortened life spans, cardiovasculardiseases (with the use chemotherapeutic agents like tamoxifen andherceptin) and vascular occlusion (stroke risk withhormonal/birthcontrol use), organ toxicity, hyperglycemia and diabetesexacerbation (with hormonal/birthcontrol use), steroidal glaucoma,hypertension (from birth control use or hormone use), and increasedsusceptibility to infections (from steroid akaloids andchemotherapeutics agents) or other types of cancers. In this manner,unwanted side effects which may be associated with certain members ofsteroidal aklyloids can be reduced with the method compounds. Forexample, using the drug screening assays described herein, and theapplication of combinationalorial and medicinal chemistry techniques,provides a means for identification of individual agents best suited forreducing the unwanted negative side effects of other actives that ispart of this application.

Dosages of C₁₉H₁₉N₇O₆ can vary from about 100 μg to about 2 mg per day,preferably at least about 200 μg per day, more preferably at least 400μg per day and should preferably be no more than about 1.6 mg per day,more preferably not more than about 1.2 mg per day. Specific pre-nataldosages of folic acid are well known and any of the literature dosagesof this component will be suitable, especially 0.4 mg, 0.6 mg, 0.8 mg,1.0 mg, 1.2 mg, and 1.4 mg for example. Other folate sources beyondC₁₉H₁₉N₇O can be used with or instead of C₁₉H₁₉N₇O₆ in amounts thatappropriate to result in the same C₁₉H₁₉N₇O₆ delivery as theaforementioned folic acid. Combinations of C₁₉H₁₉N₇O₆ and other folatesources are administered in appropriate amounts so that the total isequivalent to a C₁₉H₁₉N₆O₆ dose within the above limitations.

D-Chiroinositol doses (and the various derivatives thereof calculated onthe basis of unphosphorylated D-chiroinositol) range from about 0.05mg/day to about 60 grams per day, preferably about 0.05 mg/day to about30 grams per day, preferably about 0.1 mg to about 25 grams/day, morepreferably, about 1 mg to about 20 grams/day, still more preferablyabout 5 mg to about 10 grams per day, even more preferably about 10 mgto about 5 grams per day, yet more preferably about 25 mg to about 2grams/day, still even more preferably about 20 mg to about 1.8grams/day. Highly preferred dosages of D-chiroinositol (and its P, PP,and PolyP derivatives) further include, about 10 mg/kg/day to about 500mg/kg/day; about 100 mg to about 1 gram/day; about 1.2 gram to about 1.8gram/day; about 500 mg/day; about 500 to about 700 mg/day; about 25mg/kg/day to about 100 mg/kg/day. Particular daily doses (based onunphosphorylated D-chiroinositol) include: about 0.1 mg, about 0.2 mg,about 0.5 mg, about 0.8 mg, about 1 mg, about 1.25 mg, about 1.5 mg,about 2 mg, about 2.5 mg, about 3 mg, about 5 mg, about 10 mg, about12.5 mg, about 15 mg, about 20 mg, about 25 mg, about 40 mg, about 50mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 200 mg,about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 500) mg,about 750 mg, about 800 mg, about 1 g, about 1.2 g, about 1.4 g, about1.6 g, about 1.8 g, about 2 g, about 2.4 g, about 2.5 g, about 2.75 g,about 3 g, about 3.5 g, about 4 g, about 5 g, about 6 g, about 8 g,about 10 g, about 12 g, about 15 g, about 18 g, about 20 g, about 22.5g, about 25 g, about 30 g, about 40 g, about 50 g and about 60 g. These,particularly the larger doses, may be administered in fractional doses,all at a single time or spread out over the day as may be convenient.

In another aspect, the present invention provides pharmaceuticalpreparations comprising the subject compounds. The compounds for use inthe subject method may be conveniently formulated for administrationwith a biologically acceptable and/or sterile medium, such as water,buffered saline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like) or suitable mixtures thereof. Theoptimum concentration of the active ingredient(s) in the chosen mediumcan be determined empirically, according to procedures well known tomedicinal chemists. As used herein, “biologically acceptable medium”includes any and all solvents, dispersion media, and the like which maybe appropriate for the desired route of administration of thepharmaceutical preparation. The use of such media for pharmaceuticallyactive substances is known in the art. Except insofar as anyconventional media or agent is incompatible with the activity of thesubject compounds, its use in the pharmaceutical preparation of theinvention is contemplated. Suitable vehicles and their formulationinclusive of other proteins are described, for example, in Remington'sPharmaceutical Sciences (Mack Publishing Company, Easton, Pa., USA1985). These vehicles include injectable “deposit formulations”.

The composition which is applicable to all aspects of the presentinvention is provided in various forms and formulations, and includeswithout limitation, as an implant, a topical and transdermalformulation, as a slow release formulation, as an inhalable andvaporizable composition, and in injectable form among others. Thecomposition may be part of a kit, along with instructions for theadministration of the therapeutic agent, and optionally syringe(s) andneedles, an inhalant device, a transdermal device, and the like.

In addition to forms set out earlier, the agent may be administered inthe form of a solid, such as tablets, dragees, capsules, powders,suppositories, etc., and as a solution, suspension, or emulsion in acarrier. Particularly desirable are formulations for systemic andtopical administration, e.g., oral, injectable, topical, transdermal,including those for iontophoretical delivery, implantable, and vaginal,rectal, intranasal, intrapulmonary, and other types of formulations,which may be prepared by methods known in the art. Solid and liquidcarriers are suitable, and are known in the art. Liquid carrierstypically used in preparing solutions, suspensions, and emulsions, whichare contemplated for use in the practice of the present inventioninclude water, salt solutions, such as saline, pharmaceuticallyacceptable organic solvent(s) and their mixtures, pharmaceuticallyacceptable oils or fats, and mixtures of any and all of the above. Thecarrier may contain other therapeutic agents and suitablepharmaceutically acceptable additives such as solubilizers, emulsifiers,nutrients, buffers, preservatives, suspending agents, thickening agents,viscosity regulators, and stabilizers, among others. Suitable organicsolvents include, for example, monohydric alcohols, such as ethanol, andpolyhydric alcohols, such as glycols. Suitable oils include, forexample, soybean oil, coconut oil, olive oil, safflower oil, cottonseedoil, and the like. For parenteral administration, the carrier may alsobe an oily ester such as ethyl oleate, isopropyl myristate, and thelike. Other pharmaceutically acceptable forms include microparticles,microcapsules, liposomal encapsulates, and the like, as well as theircombinations of the agent of the invention, alone or with one or moreother therapeutic agents may be formulated into sustained releasemicroparticles or microcapsules. Materials suitable for themicroparticle matrix include materials such as starch, polyvinylalcohol, polyvinylpyrrolidinone, polyacrylic acid, and the like, as wellas combinations of any two or more thereof. Biodegradable polymerssuitable for use as a microparticle or microcapsule matrix include,without limitation, for example, poly-1-lactide, poly-dl-lactide,polyglycolide, poly(glycolide-co-dl-lactide), polyanhydrides,polyorthoesters, poly(alpha-hydroxybutyric acid), poly-p-dioxinone, andblock polymers of polyglycolide, trimethylene carbonate, polyethyleneoxide, proteins, polysaccharides, and derivatives and mixtures thereof.The microparticles and microcapsules containing the agent may beprepared employing methods which are well known in the art, e.g., bysolvent evaporation, phase separation, and interphase reaction methods,spray drying, physical methods, and the like. As already indicated, thepresent agent may also be encapsulated into liposomes, and themicroparticles, microcapsules, and/or liposomes loaded with the agentmay then be suspended or emulsified in a suitable liquid carrier.

In addition to the manners set forth above, any aspect of the presentagent may be administered in a variety of ways, including topical,enteral, and parenteral routes of administration. For example, withoutlimitation, suitable modes of administration include subcutaneous,transdermal, transmucosal, including iontophoretic, intravenous,subcutaneous, transnasal, intrapulmonary, transdermal, oral, rectal,vaginal, implantable and the like, as well as their combinations. Theparticular pharmaceutically acceptable firm of the therapeutic agentemployed will depend on the route of administration selected. The agentmay be, for example, administered in a form that enhances itsbioavailability when compared with standard oral formulations. Suitableforms include a lipid carrier system that promotes the oral absorptionof compounds through the intestinal epithelium. Examples of thesesystems are oil-in, water, and water-in-oil emulsions. Exemplary oilsthat are contemplated for use in oil-in-water and water-in-oil basedsystems include castor oil, olive oil, soybean oil, safflower oil,coconut oil, cottonseed oil, their combinations, and the like. Othersuitable forms that enhance the bioavailability of the orallyadministered agent of this invention include single surfactant, andmixed micelle systems. The agent may, for example, be orallyadministered in the form of a mixed micelle system containing linoleicacid and polyoxyethylene-hardened castor oil. Suitable surfactantscontemplated for use in single and mixed micelle systems includepolyoxyethylene ether, polyoxypropylene ether, polyoxyethylene lauryl,cetyl and cholesteryl ethers, polyoxyethylene derivatives of lanolinalcohols, and the like, as well as their mixtures.

When intravenous or subcutaneous administration is contemplated, the useof a solution of the therapeutic agent is preferred. For transdermaladministration by iontophoresis, the agent is preferably administered incharged form, such as in the form of a salt. The salt may be in solutionor in a gel reservoir. Therapeutic agent-containing gels may be used asa drug reservoir for many routes of administration. An agent containinggel may be prepared by blending the inositol based compound with ahydrogel-forming polymer such as polyvinyl alcohol, polyacrylamides,copolymers of propylene oxide and ethylene oxide, e.g., Pluronic®,polyvinylpyrrolidinone, gelatin, polymers and copolymers of maleicanhydride, polyacrylic acid and salts and derivatives thereof,polysaccharides, and salts and derivatives thereof, cellulosic polymers,and salts and derivatives thereof, polycarboxylic acids, and the like,as well as their mixtures. The agent may also be administeredtransdermally through the use of a skin patch, with a carrier. Suitablecarriers are typically inert to the agent, non-toxic to the skin, andallow the delivery of the agent for systemic absorption into the bloodstream via the skin. Carriers for transdermal absorption may includepastes, such as absorptive powders dispersed in petroleum or hydrophilicpetroleum with the agent, with or without a carrier, or a matrixcontaining inositol. Preparations of agent-based compounds may also beadministered topically as a solution, cream, lotion, or gel, formulatedwith pharmaceutically acceptable vehicles containing the agent. Theagent may also be administered intra or transnasally or intrapulmonarilyas an aerosol spray of a solution, suspension or emulsion, or asmicroparticles, microcapsules, or liposomes containing the agent. Alsocontemplated are formulations of the agent of this invention withpharmaceutically acceptable excipients. Suitable excipients contemplatedfor use as processing aids and drug delivery modifiers and enhancersinclude calcium phosphate, magnesium stearate, talc, monosaccharides,disaccharides, starch, gelatin, cellulose, methyl cellulose, sodiumcarboxymethyl cellulose, dextrose, polyvinylpyrrolidinone, low meltingwaxes, ion exchange resins, and the like, as well as their combinations.Typically, such formulations are prepared as tablets or capsules. Otherformulations are, however, also within those contemplated in thispatent.

The agent may be administered as a single dose or in multiple doses.Multiple doses may be administered either continuously, in intervals, ora combination of both. The agent, for example, may be administered as asingle dose, optionally coupled with a follow-up dose. The follow-updose may be administered by the same or different route ofadministration as a single or sustained dose. Accordingly, the presentcomposition is presented in unit dosage form or in multiple dosage form,as well as in the form of a kit, which may be for self administration,along with instructions for the use of the therapeutic agent, andoptionally a syringe(s) and needle(s), an inhaler or vaporizer, atransdermal patch, optionally for iontophoresis, and the like. Thecomposition is also provided as a cream or gel for topical application,and as an implant. The manufacture of implants is known in the art andcommercially available.

One particular form of implantable version of the present invention isthe use of a dendrimeric type of carrier, which may have the inositolbased compound attached thereto by an ionic, covalent, or hydrogenbonding. Alternatively, the inositol component of the invention bound toa fibroblast bound dendrimer (which fibroblast will anchor the deliverysystem in place) is also contemplated. Dendritic molecules have multipleprotrusions which may also be used to attach the dendrimer to aparticular site within the body, to particular cells, or can beimplanted for migration within the body for attachment to particularcites at some other point in time. (See for example Dendrimers ImproveCancer Drug Uptake and Antitumor Activity, Drug Delivery, BostonUniversity, NCI Alliance for Nanotechnology in Cancer—NanoTechWire_com—The online resource for Nano Technology And Research, Jan. 15, 2007available at http://nanotechwire.com/news.asp?nid=4213. Alternatively,the protrusions themselves may have the inositol component bound theretoor coated thereon.

Another implantable version is the use of nanobots or nanorobots for thedelivery of the active agent. While nanorobotics is a rather recentdevelopment, those of ordinary skill will appreciate the advantages ofsuch a delivery, which can be achieved in manners set forth in forexample (a) Shanthi, et al; Prospects for medical Robots; AZojonoJournal of Nanotechnology Online, posted 13 Nov. 2007;

http://www.azonano.com/Details.asp?ArticleID═2035; (b) AdrianoCavalcanti, Bijan Shirinzadeh, Tad Hogg, Julian A. Smith, “HardwareArchitecture for Nanorobot Application in Cancer Therapy”, IEEE-RAS ICARInt'l Conf. on Advanced Robotics, Jeju, Korea, pp. 200-205, August 2007;(c) Hede et al, “Nano”: the new nemesis of Cancer, J Can Res Ther[serial online] 2006 [cited 2007 Dec. 11]; 2:186-195 available fromhttp://www.cancerjournal.net/text.asp?2006/2/4/186/29829 and (d)Nanomrobotics Control Design and 3D Simulation, available athttp://www.nanorobotdesign.com/_(2007). A preferred version of thenanorobotic delivery in the present invention is designed to beimplanted in or around the site of specific delivery such as a cancerouslesion excision site or into an inoperable tumor and which delivers theactive agent or active agent precursor on a single prolonged or multiplerelease schedule which can be pre-programmed for delivery over short orextended periods extending for as much as multiple years. As may bedesired, the nanorobotic delivery system can be one which can mirate orbe fixed in position by virtue of specific adherence mechanismsincluding fibroblasts, monoclonal antibodies, charged particle portions,antisence DNA, etc.

Yet another implantable or injectable and migratable delivery systemutilizes monoclonal antibodies that are specific to cancer cellreceptors or other cancer cell specific proteins. These antibodieshaving the active agent linked thereto migrate to the specific cancercells and deliver the active agent directly to the cell on which it isto act.

Pharmaceutical formulations of the present invention can also includeveterinary compositions, e.g., pharmaceutical preparations of thesubject compounds suitable for veterinary uses, e.g., for the treatmentof live stock or domestic animals, e.g., dogs.

Compositions of the present invention may be single active agententities that are merely co-administered as described herein or fixedcombinations as indicated above. The other components beyond the folicacid (and/or other folate) and the inositol compounds (and/or P, PP,and/or PolyP derivative thereof, and the other derivatives thereofdiscussed further herein) can be selected from a wide variety ofcompounds. Additional active agents that may be included in or merelyco-administered with the above components include those estrogenic andprogestogenic substances used in birth control pills, hormonereplacement therapy, androgen ablative therapy, etc. (including, but notlimited to conjugated estrogens, ethinyl estradiol, levonorgestrel,norgestrel, norgestimate, norethidrone, norethidrone acetate, mestranol,ethynodiol diacetate, norelgestromin, etonogestrel, desogestrel, etc).These hormones are currently marketed under the following (non-limiting)trade names: ALESSE, ANGELIQ, DIANE, LEVLEN, LO-OVRAL, LYBREL,TRICYCLEN, ORTHOCEPT, ORTHOEVRA, MIRENA, MENOSTAR, NUVA RING, OVRAL,TRI-LEVLEN, TRIPHASIL, BREVICON, FEMHRT, LOESTRIN, LoOGESTREL,MICROGESTIN, YAZMIN, among others. Where the birth control or hormonereplacement therapy dosage form is other than an oral dosage form (suchas, for example, a transdermal patch (in the case of currently marketednorelgestromin) or a vaginal ring (in the case of currently marketedetonogestrel estradiol), ORTHOEVRA marketed as transdermal birthcontrolpatch (recently linked to higher than average acute thromboemboliticevents in female users). The invention compound is designed to eliminatethe inherent risk of this type of hormonal contraception. Alsotransdermal patches for hormonal replacement including but not limitedto Vivelle®and Vivelle-Dot®, Estradot®, combination estrogen/progestintransdermal delivery systems (including CombiPatch®, licensed toAventis, and Estalisc®, Testoderm®. The invention objectives areachieved with a co-therapy of a suitable dosage form with or without thefolic acid (and/or other folate source) and D-chiroinositol (and/or P,PP, and/or Poly P derivatives thereof). Andogen ablative therapies forwhich the instant invention can be used include treatment with forexample, without limitation, finasteride as well as other known androgenablative drugs. Other active compounds for use in combination with or incotherapy with the inositol compounds of the invention include,especially in the fetal alcohol syndrome prevention aspect, those usedin cholesterol reduction regimens, especially the statins.

Compositions of the present invention in which the D-chiroinositol orother inositol component and or the folic acid component are the onlyactive agents can be prepared as in or analogously to those set forth inthe patents indicated above as being incorporated herein by reference.For compositions that are disclosed therein that have an inositolcomponent, the D-chiroinositol or other inositol component (and/or P,PP, and/or PolyP derivative thereof or other derivative thereofdiscussed further herein) can be used in direct replacement of the suchinositol component indicated in such reference. The folic acid (and/orother folate source) can be incorporated therein by merely replacing asmall portion of filler or merely adding the folic acid (and/or otherfolate source) thereto. Where the referenced formulation is a folic acidformulation and the dose selected for the inositol (and/or P, PP, and/orPolyP derivative thereof or other derivative thereof discussed furtherherein) is sufficiently small, the inositol (and/or derivative thereof)can be used in place of a portion or all of the filler used in thereferenced formulation, or added to it. If larger amounts are needed,then the filler used in the referenced formulation is replaced with theinositol (and/or P, PP, and/or PolyP derivative thereof or otherderivative thereof) component if the resulting tablet size is not ofconcern. If the size of the dosage form is insufficient to accommodatethe full dose of the inositol component, then either a separate dosageform is used or multiple dosage forms having a fraction of the dailydose is used and the patient will need to take more than 1 dosage formto achieve the daily dosages set forth.

In preferred dosage forms of one embodiment of the invention, theD-chiroinositol (and/or P, PP, and/or PolyP derivatives thereof or otherderivatives thereof) is substantially free of the other isomers ofinositol. In highly preferred formulations of this embodiment, theD-chiroinositol (and/or the P, PP, and/or PolyP derivatives thereof orother derivatives thereof) and the dosage forms thereof are completelyfree of the other isomers of inositol as well as their correspondingphosphorylated derivatives. For purposes of the present invention,“substantially free” means not more than about 5% based on the combinedD-chiro forms present, more preferably not more than about 2.5%, stillmore preferably not more than about 1%, most preferably not more than0.5%. For purposes of the present invention, “completely free of” or“free of” means below the limit of detection of said non-D-chiro formsrespectively in common analytical techniques used in commonpharmaceutical quality control of bulk materials as of the date of theinvention herein. Similarly with respect to formulations of otherembodiments of the invention in which other inositol isomers, theirrespective phosphorylates (of varying size) or other derivatives thereofas described further herein, preferred are those that are substantiallyfree of other isomeric forms of inositol than the one being primarilypresent, and more preferably completely free of such other isomericforms.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

The therapeutic agent of this aspect of the invention (thenon-D-chiroinositol based inositol compounds), is typically administeredto a subject at a dose of about 20 to about 8,000 mg/kg/day, andpreferably at about 30 to about 5,000 mg/kg/day. Other amounts, however,may also be administered. Higher or lower doses of these agents,however, may also be administered.

U.S. Pat. No. 5,998,485 (incorporated herein by reference) reports thatunmodified scyllo inositol is more potent than other unmodified inositolisomers tested there in modulating a subject's immune response in a dosedependent manner. When the agent is administered at relatively lowdoses, it effectively enhances a mammal's immune response and, whenadministered at relatively high doses, inositol effectively inhibits orsuppresses the mammal's immune response. Thus, the gain-of-function orloss-of-function activity of the inositols may vary depending on doseand the above screenings should be undertaken at multiple dosingschedules to determine whether an analogous result is present withrespect to other inositol based compounds within the present invention,and if so, the compound may be used in the appropriate indication aboveat the appropriate dose NOTWITHSTANDING prior direction to not use anyparticular active agent in such indication.

The dose response range for gain-of-function as opposed toloss-of-function for any particular pathway may vary somewhat dependingon the form of the agent employed, such as, the particular stereoisomer,derivative, or salt employed. One of ordinary skill in the art, however,may readily determine the range of response-enhancing doses of thetherapeutic agent by the above screening tests and by other means knownin the art, e.g., by generating a dose-response curve for any particularform of therapeutic agent. The dose of the therapeutic agentadministered for any particular activity will, of course, also vary withfactors such as the pharmacodynamic characteristics of the agentemployed, its mode and route of administration, the age, health, andweight of the recipient, the nature and extent of the symptoms, the kindof concurrent treatment(s), the frequency of treatment, the effectdesired, and the like. Modulation of the doses within the ranges setforth herein are within the skill of the ordinary skilled clinician andcan be adjusted by such persons appropriately.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient will range from about 0.0001 to about 100 mg perkilogram of body weight per day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacious biopharmaceuticals. A variety ofpolymers (including hydrogels), can be used to form an implant for thesustained release of a subject compound at a particular target site.Other methods of drug delivery may also be provided by nanotechnologyutilizing such nanomaterials, nanostructures, nanofibers, nanowires,nanoparticles, quantum dot, nanotube, demdrimer, nanocystal, or nanobot.(See HIGH-PRESSURE POLYMERIZATION OF SINGLE WALL CARBON NANOTUBES; M.Popov, M. Kyotani and Y. Koga Joint Research Consortium of FrontierCarbon Technology, JFCC, c/o NIMC, Higashi, Tsukuba, Ibaraki, 305-8565,Japan and R. J. Nemanich Department of Physics, North Carolina StateUniversity, 408A Cox, Box 8202, Raleigh, N.C., 27695-8202, USA;http://www.eng.aubum.eduldepartmenl/ee/ADC-FCT2001/ADCFCTabstract/101.htm)

Other methods of entry may also be provided by a battery. For example, abattery operated transdermal drug delivery device (iontophoresis)utilizing a current distribution member for delivering a pulsed directcurrent sufficient to iontophoretically deliver composition across astratum corneum layer of the epidermis. The current distribution membercomprises an electrochemically active component in electrical connectionwith a battery, a voltage pulse generator and a precision resistor.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by implant, injection,inhalation, eye lotion, ointment, drops, suppository, controlled releasepatch, etc. administration by injection, infusion or inhalation; topicalby lotion or ointment; and rectal by suppositories. Oral and topicaladministrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intrasystemically, and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms such as described below orby other conventional methods known to those of skill in the art.

The term “treatment” is intended to encompass prophylaxis, therapy andcure, unless specifically indicated otherwise or the context requiresotherwise.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable and/or sterile carriers andcan also be administered in conjunction with other antimicrobial agentssuch as penicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

III. Pharmaceutical Compositions

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). The subject compoundsaccording to the invention may be formulated for administration in anyconvenient way for use in human or veterinary medicine. In certainembodiments, the compound included in the pharmaceutical preparation maybe active itself, or may be a prodrug, e.g., capable of being convertedto an active compound in a physiological setting.

Thus, another aspect of the present invention provides pharmaceuticallyacceptable compositions comprising a therapeutically effective amount ofone or more of the compounds described herein, formulated together withone or more pharmaceutically acceptable carriers (additives) and/ordiluents. As described in detail below, the pharmaceutical compositionsof the present invention may be specially formulated for administrationin solid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, pastesfor application to the tongue: (2) parenteral administration, forexample, by subcutaneous, intramuscular or intravenous injection as, forexample, a sterile solution or suspension; (3) topical application, forexample, as a cream, ointment or spray applied to the skin; or (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam. However, in certain embodiments the subject compounds may besimply dissolved or suspended in sterile water. In certain embodiments,the pharmaceutical preparation is non-pyrogenic, i.e., does not elevatethe body temperature of a patient.

The phrase “therapeutically effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect, e.g., with respect to D-chiroinositol and itsderivatives by overcoming a ptc loss-of-function, hedgehoggain-of-function, or smoothened gain-of-function, in at least asub-population of cells in an animal and thereby blocking the biologicalconsequences of that pathway in the treated cells, at a reasonablebenefit/risk ratio applicable to any medical treatment.

The phrase pharmaceutically acceptable is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Such materials willbe known to those of ordinary skill in the pharmaceutical formulationart.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject regulatorsfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient Some examples of materials which can serve aspharmaceutically acceptable carriers include without limitation: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;(4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar, (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water. (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions;and (21) other non-toxic compatible substances employed inpharmaceutical formulations.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable acids. The term “pharmaceutically acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ during the final isolation andpurification of the compounds of the invention, or by separatelyreacting a purified compound of the invention in its free base form witha suitable organic or inorganic acid, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

As described above, where the compounds used in the invention arecapable of salt formation, the reference to the compound includes thepharmaceutically acceptable salts thereof. These salts can be preparedin situ during the final isolation and purification of the compounds ofthe invention, or by separately reacting a purified compound of theinvention in its free form with a suitable organic or inorganic acid orbase as appropriate, and isolating the salt thus formed, or reacting asalt of the compound with an appropriate organic or inorganic acid orbase to result in a different salt formation. Representativepharmaceutically acceptable acid addition salts include, withoutlimitation, the hydrobromide, hydrochloride, sulfate, bisulfate,phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate,laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate,fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate,lactobionate, and laurylsulphonate salts and the like. (See, forexample, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci.66:1-19).

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. (See, forexample, Berge et al., supra).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, stearic acid, and talc, as well ascoloring agents, release agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral ortopical (including buccal and sublingual), rectal, vaginal, and/orparenteral, transdermal, iontophoresis, nano particle delivery (withoutlimitation), and various polypeptide vectors “carrier” administrationfrom one organ to another as necessary. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will vary depending upon the host being treated, theparticular mode of administration. The amount of active ingredient whichcan be combined with a carrier material to produce a single dosage formwill generally be that amount of the compound which produces atherapeutic effect.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product. Alternative methods will be appreciatedby those of ordinary skill in the pharmaceutical formulating art.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (usually using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as, without limitation, sodium citrate or dicalciumphosphate, and/or any of the following: (1) fillers or extenders, suchas starches, lactose, sucrose, glucose, mannitol, and/or silicic acid;(2) binders, such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants,such as glycerol; disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate; (5) solution retarding agents, such as paraffin;(6) absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay, (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent, etc. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the subject compounds inthe proper medium. Absorption enhancers can also be used to increase theflux of the compound across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe compound in a polymer matrix or gel or microneedle technology.Battery operated trandermal drug delivery utilizing a current is anotherexample of controlling rate of flux (Iontophoretic transdermal delivery)

Ophthalmic formulations, ophthalmalic eye implant for medicationdelivery, eye ointments, drug eluting contact lenses, powders, solutionsand the like, are also contemplated as being within the scope of thisinvention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, s olutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline, or nanocrystal, or amorphous material having poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in polymers such as, without limitation,polylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations arealso prepared by entrapping the drug in liposomes or microemulsionswhich are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”, W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feeds andFeeding” O and B books, Corvallis, Oreg., U.S.A., 1977).

Synthetic schemes using coupling reactions to identity active regulatorsof genes or gene expression.

The subject compounds, and derivatives thereof, can be prepared readilyby employing synthetic methodology well-known in the art. Additionalcompounds may be synthesized and tested in a combinatorial fashion, tofacilitate the identification of additional compounds which may beemployed in the subject method.

A. Combinatorial Libraries

The compounds of the present invention, particularly libraries ofvariants having various representative classes of substituents, areamenable to combinatorial chemistry and other parallel synthesis schemes(see, for example, PCT WO 94/08051). The result is that large librariesof related compounds, e.g. a variegated library of compounds representedabove, can be screened rapidly in high throughput assays in order toidentify potential hedgehog regulator lead compounds, as well as torefine the specificity, toxicity, and/or cytotoxio-kinetic profile of alead compound. For instance, ptc, hedgehog, or smoothened bioactivityassays, such as may be developed using cells with either a ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function, can be used to screen a library of the subjectcompounds for those having agonist activity toward ptc or antagonistactivity towards hedgehog or smoothened. Alternatively, bioactivityassays using cells with either a ptc gain-of-function, hedgehogloss-of-function, or smoothened loss-of-function, can be used to screena library of the subject compounds for those having antagonist activitytoward ptc or agonist activity towards hedgehog or smoothened.

Simply for illustration, a combinatorial library for the purposes of thepresent invention is a mixture of chemically related compounds which maybe screened together for a desired property. The preparation of manyrelated compounds in a single reaction greatly reduces and simplifiesthe number of screening processes which need to be carried out.Screening for the appropriate physical properties can be done byconventional methods.

Diversity in the library can be created at a variety of differentlevels. For instance, the substrate aryl groups used in thecombinatorial reactions can be diverse in terms of the core aryl moiety,e.g., a variegation in terms of the ring structure, and/or can be variedwith respect to the other substituents.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules such as the subjectcompounds. See, for example, Blondelle et al. (1995) Trends Anal. Chem.14:83; the Affymax U.S. Pat. Nos. 5,359,115 and 5,362,899: the EllmanU.S. Pat. No. 5,288,514: the Still et al. PCT publication WO 94/08051;the ArQule U.S. Pat. Nos. 5,736,412 and 5,712,171; Chen et al. (1994)JACS 116:2661: Kerr et al. (1993) JACS 115:252; PCT publicationsWO92/10092, WO93/09668 and WO91/07087; and the Lerner et al. PCTpublication WO93/20242). Accordingly, a variety of libraries on theorder of about 100 to 1,000,000 or more diversomers of the subjectcompounds can be synthesized and screened for particular activity orproperty.

Combinatorial library development and screening can be conducted, forexample in analogy to the methods and procedures set forth in Beachy etal U.S. Pat. No. 7,291,626, incorporated herein by reference in itsentirety. In an exemplary embodiment, a library of candidate compounddiversomers can be synthesized utilizing a scheme adapted to thetechniques described in the Still et al. PCT publication WO 94/08051,e.g., being linked to a polymer bead by a hydrolyzable or photolyzablegroup, optionally located at one of the positions of the candidateregulators or a substituent of a synthetic intermediate. According tothe Still et al. technique, the library is synthesized on a set ofbeads, each bead including a set of tags identifying the particulardiversomer on that bead. The bead library can then be “plated” with, forexample, ptc loss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function cells for which a hedgehog antagonist is sought. Thediversomers can be released from the bead, e.g. by hydrolysis.

Many variations on the above and related pathways permit the synthesisof widely diverse libraries of compounds which may be tested asregulators of hedgehog function.

B. Screening Assays

There are a variety of assays available for determining the ability of acompound such as a hedgehog regulator to regulate ptc, smoothened, orhedgehog function, many of which can be disposed in high-throughputformats. In many drug screening programs which test libraries ofcompounds and natural extracts, high throughput assays are desirable inorder to maximize the number of compounds surveyed in a given period oftime. Thus, libraries of synthetic and natural products can be sampledfor other compounds which are hedgehog regulators. Such assays can beconducted by analogy described below by Beachy et al U.S. Pat. No.7,291,626, (2007).

In addition to cell-free assays, test compounds can also be tested incell-based assays. In one embodiment, cells which have a ptcloss-of-function, hedgehog gain-of-function, loss of kinase activity,smoothened gain-of-function phenotype can be contacted with a test agentof interest, with the assay scoring for, e.g., inhibition ofproliferation of the cell in the presence of the test agent.

A number of gene products have been implicated in receptor-mediatedsignal transductions, including patched, GL1, GL2, GL3 family oftranscription the serine/threonine kinase fused (fu) and smoothened, andpatched and the induction of cells by hedgehog proteins sets in motion acascade involving the activation and inhibition of downstream effectors,the ultimate consequence of which is, in some instances, a detectablechange in the transcription or translation of a gene. Potentialtranscriptional targets of hedgehog-mediated signaling are described.

Reporter gene based assays described in the invention by Beachy et al,2007 can be utilized by analogy for the subject compositions in the sameway. These gene based assays appear to measure the end stage of theabove described cascade of events, e.g., transcriptional modulation.

Accordingly, in practicing one embodiment of the assay, a reporter geneconstruct is inserted into the reagent cell in order to generate adetection signal dependent on ptc loss-of-function, hedgehoggain-of-function, smoothened gain-of-function, or stimulation by Shhitself or inhibitions of protein kinases. The amount of transcriptionfrom the reporter gene may be measured using any method known to thoseof skill in the art to be suitable. For example, mRNA expression fromthe reporter gene may be detected using RNAse protection or RNA-basedPCR, or the protein product of the reporter gene may be identified by acharacteristic stain or an intrinsic biological activity. The amount ofexpression from the reporter gene is then compared to the amount ofexpression in either the same cell in the absence of the test compoundor it may be compared with the amount of transcription in asubstantially identical cell that lacks the target receptor protein. Anystatistically or otherwise significant decrease in the amount oftranscription indicates that the test compound has in some manneragonized the normal ptc signal (or modulated, antagonized thegain-of-function hedgehog or smoothened signal), e.g., the test compoundis a potential hedgehog antagonist.

C. Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXAMPLES

The following non-limiting examples are presented only to exemplifyvarious embodiments of the invention and do not limit it in any fashion.

Example 1

In Jenkins, D. at al Anat 2007 Sep. 11, it states that studies of mousemutants have demonstrated that Sonic hedgehog (SHH) signalling has afunctional role in morphogenesis and differentiation at multiple siteswithin the forming urinary tract, and urinary tract malformations havebeen reported in humans with mutations that disrupt SHH signalling.However, there is only strikingly sparse and fragmentary informationabout the expression of SHH and associated signalling genes in normalhuman urinary tract development. (Jenkins, 2007) usedimmunohistochemistry to demonstrate that SHH protein was localised indistinct urinary tract epithelia in developing normal humans, in theurothelium of the nascent bladder and in kidney medullary collectingducts. The expression patterns of the SHH-transducing proteins Patched(PTCH) and Smoothened (SMO) were consistent with long-range paracrinesignalling associated with detrusor smooth muscle differentiation in theurogenital sinus. In the developing kidney, SHH and PTCH were expressedin epithelia of the collecting system between 16-26 weeks—surprisingly,SMO was not detected. Analysis of cell proliferation and Cyclin B1immunohistochemistry at 26 weeks, as compared with a 28 week sample inwhich SHH expression was down-regulated, was consistent with the ideathat SHH and PTCH might influence medullary collecting duct growth byregulating the subcellular localisation of Cyclin B1 independently ofSMO.

Collectively, this descriptive experimental result is similar to that ofBeachy et al, 2007. These results, along with the inventors ownliterature research, certainly generates new hypotheses and evidenceregarding SHH signal transduction in human urinary tract development aswell as other anomalies described in the patent that help to explain thevaried urinary tract malformations associated with anorectalmalformation phenotypes noted in individuals with mutations in the SHHpathway.

Example 2

Females determined to be at risk of fetal malformations and who areseeking a further pregnancy are split into no treatment, folatetreatment, D-chiroinositol treatment, and Folate+D-chiroinositoltreatment arms. The respective regimens are administered once daily frombefore conception through the end of the first trimester. Relative tothe untreated controls, the frequency of fetal malformations is reducedin each of the non-control arms. However, the reduction in frequency offetal malformations in the co-therapy of the present invention issignificantly better than in either of the other treatment arms.

Example 3

Females beginning birth control medication are assigned to similartreatment and control groups as in Example 1. Treatment is begun at thetime of initiation of birth control medication, and continued untilafter a pregnancy occurs and for the following first trimester ofpregnancy. Similar reductions as reported in Example 2 are seen. Inaddition, follow up of these females shows a lower level of breastcancer development than expected.

Example 4

Men preparing to initiate androgen ablative therapy are initiated on acourse of D-chiroinositol prior to and throughout the treatment with theandrogen ablative therapeutic. The frequency of male breast cancer foundin these patients is substantially reduced as compared to controls notreceiving the D-chiroinositol therapy.

Example 5

In order to demonstrate an effect of D-chiroinositol plus folate on Shhsignaling during embryogenesis, curly tail mice (38) are chosen becauseof the genetic propensity for spinal defects and associatedgenitourinary (GU) defects. These embryos are exposed to the presentinvention compound D-chiroinositol vs. myoinositol and internalexamination is conducted. The severity of these defects among treatedembryos treated with myoinositol are severe compared to those embryostreated with D-chiroinositol.

Example 6

To determine whether the downstream targets of the phosphatidyl inositol(PI3K) signaling pathway could be effected by an inhibitor, we can use avehicle to inhibit the effect of inositol on neural tube closure. Thecurly tail mice embryos are cultured from E9.5 to E10.5, and the lengthof unclosed neural folds at the pnp are measured to indicatepredisposition to spina bifida.

Embryos which are exposed to the kinase inhibitor alone have enlargedPNP's, reflecting the in vivo development of spinal NTD's by 50%-60% ofcurly tail embryos. Hence, inhibition of the down stream targets of thephosptidylinositol signaling pathway blocks the protective effect of theinositol. Therefore, D-chiroinositol normalizes PNP closure in vitro.

D-chiroinositol, a phosphate derivative, plus folic acid could representa possible novel adjunct therapy to prevent NTD's because the activationof certain isoforms by the phostidylinositol pathway is essential forprevention of these defects.

It is well-known that cancer remains a major health problem in theUnited States and in other developed countries. It is our effort here toreduce the burden of cancer on a physical, social, and psychologicallevel. There is a constant search for more effective cancer treatments.While, it well-known that many cancer specialists have won the battlefor detecting and treating many cancers, however, there is still oneproblem that remains; the concept of prevention. This is a promisingapproach to control cancer. Also, the discovery of convergent signalingpathways in these contexts are something that cannot be overlooked aswell because these signaling pathways control many cellular processesincluding cellular proliferation, survival, growth, and motility, all ofwhich are critical processes for tumorigenesis. Without being bound totheory, it is the inventor's belief that the alteration of theseconverged pathways occurs in many cancerous states, many embryonicdysmorphic states, as well as many conditions that are the result ofaberrant signaling, but otherwise unrelated to each other.

Based on the foregoing and the results described below by Beachy et al,2007, the inventor suggests that the inclusion of the inositol compound,alone or in combination with other compounds as set forth more fullyherein, is warranted as a treatment or prevention for a wide range ofconditions related to aberrant growth (such as for cancers) birthdefects due to pattern formation dysregulation during gestation, as wellas other chronic diseases.

The goal is to identify combinations of the invention that target thetumor at vulnerable sites and interrupt specific pathways suspected incarcinogenesis. From the behavior and characteristics of malignantcells, several principal pathways of malignancy have been established.They include: Cell proliferation, cell cycle progression, metastases andinvasion, angiogenesis, and apoptosis. Interestingly, we believe that atleast one of the D-chiroinositolphosphates targets and acts on all ofthem. MDA-MB 231 human breast cancer cells are highly invasive tumorcells. These, and most other, tumors cells omit substances known asmatrix metalloproteinases that allow metastatic cells to pass into bloodvessels, myoinositolhexaphosphate significantly inhibits secretion ofMMP-9 from MDA-MB 231 cells.

Introduction

Beachy, (2007) goes on to describe an essential role for Shh during HairFollicle Morphogenesis. The hair follicle is a source of epithelial stemcells and site of origin for several types of skin tumors. While it isclear that follicles arise by way of a series of inductive tissueinteractions, identification of the signaling molecules driving thisprocess remains a major challenge in skin biology. Hair germs comprisingepidermal placodes and associated dermal condensates were detected inboth control and Shh −/− embryos, but progression through subsequentstages of follicle development was blocked in mutant skin. Theexpression of Gli1 and Ptc1 was reduced in Shh −/− dermal condensatesand they failed to evolve into hair follicle papillae, suggesting thatthe adjacent mesenchyme is a critical target for placode-derived Shh.Despite the profound inhibition of hair follicle morphogenesis,late-stage follicle differentiation markers were detected in Shh −/−skin grafts, as well as cultured vibrissa explants treated withcyclopamine to block Shh signaling. These findings are stated asrevealing an essential role for Shh during hair follicle morphogenesis,where it is required for normal advancement beyond the hair germ stageof development.

Early stages of organogenesis are marked by the appearance ofmesenchymal condensates and focal cellular aggregates, or placodes, inadjacent epithelia. This process is driven to completion by a series ofinductive signals traveling between epithelial and mesenchymal cellpopulations which ultimately give rise to the adult structure (reviewedin Gurdon, 1992; Thesleff et al., 1995). In skin appendages such asvibrissae and hair follicles, detailed analysis of tissue recombinantshas revealed the existence of at least three morphogenetic signals: theembryonic dermis instructs overlying ectoderm to initiate placodeformation; the placode transmits a signal generating a dermal condensatewith hair follicle-inductive properties; and the condensate in turnsends a signal to nascent follicle keratinocytes stimulating theirproliferation, downgrowth into the developing dermis, and reorganizationto form the mature follicle (reviewed in Sengel, 1976; Hardy, 1992). Theepithelial and mesenchymal components of the follicle remain in closeproximity in mature hair bulbs, where the dermal papilla is surroundedby matrix cells giving rise to at least six phenotypically distinctepithelial cell types in the hair shaft and inner root sheath of thehair follicle. After birth the follicle epithelium cycles throughperiods of active growth (anagen), followed by regression (catagen) andinactivity (telogen) (reviewed in Cotsarelis, 1997). The morphogeneticprogram that accompanies the transition from telogen to anagen bearssimilarities to follicle development during embryogenesis, making thisstructure a unique model for studying certain aspects of organogenesisin the adult animal. Although a, large number of genes have beenimplicated at various stages of hair follicle development and cycling(reviewed in Rosenquist and Martin, 1996; Sterm et al, 1996; Widelitz etal, 1997; Millar, 1997), the molecular nature of the inductive signalsthat underlie the formation of the follicle is largely unknown.

In situ localization of transcripts encoding potential morphogens hasrevealed focal expression of Sunic hedgehog (Shh) in placodes of theepidermis and several other epithelia at early stages of development,with Ptc1 transcripts encoding a putative Shh receptor also present inadjacent mesenchymal cells (Bitgood and McMahon, 1995; Iseki et al.,1996; Oro et al., 1997; Motoyama et al., 1998). These findings, coupledwith the accumulating evidence demonstrating a pivotal role for secretedHedgehog proteins in a variety of developmental processes (reviewed inHammerschmidt et al., 1997). Since the follicle is a source of cutaneousstem cells and a likely, site of origin for certain epithelial skincancers (Cotsarelis et al., 1990; Lavker et al., 1993; Rochat et al.,1994; Hansen and Tennant, 1994), understanding the developmental biologyof this organ is likely to provide insights relevant to normal skinfunction as well as wound-healing and neoplasia, and may shed light onfundamental aspects of organogenesis involving other structures as well.

To summarize, they concluded an obligatory role for Shh in theprogression of hair follicle morphogenesis past the hair germ stage ofdevelopment. The reduced expression of Ptc1 and Gli1 in Shh −/− dermalcondensates, coupled with their failure to evolve into recognizabledermal papillae, argue that Shh is involved in regulating development ofthe mesenchymal component of the hair follicle, although a requirementfor Shh signaling in the epithelial component of the follicle cannot beexcluded. In the absence of dermal papillae normal hair folliclemorphogenesis does not proceed, underscoring the critical influencethese cells have on growth and remodeling of developing follicleepithelium (Jahoda et al., 1984; Weinberg et al., 1993). Interestingly,biochemical differentiation of the follicle can take place in theabsence of normal morphogenesis, implying that these two processes areregulated independently in this organ. According to Beachy et al,(2007), additional experiments will be required to formally define whichcomponent of the developing follicle is functionally impaired in Shh −/−embryos, and to determine whether Shh has additional roles at laterstages of follicle development or during hair cycling. (Johnson et al.,1996; Hahn et al., 1996; Oro et al., 1997; Fan et al., 1997; Xie et al.,1998).

The experiments detailed below by Beachy et al (2007) are believed to beequally applicable to the D-chiroinositol compounds (phosphates thereofand other derivatives thereof) used in the present invention.

Methods

Animals and Skin Transplantation

The generation and identification of Shh mutant mice was performed asdescribed (Chiang et al., 1996). Embryonic skin was grafted onto thedorsal fascia of nude mice beneath a protective silicone chamber using amodification of a previously-described technique (Dlugosz et al., 1995).The chamber was removed 11-12 days after grafting and tissue harvestedfor analysis after an additional one to four weeks. Animals were handledin accordance with NIH guidelines.

Immunohistochemistry

Tissue is fixed overnight in Carnoy's or Bouin's solution for detectingkeratins (K1, K10, K5, K14, and K 17), loricrin, and filaggrin; fixationwith neutral-buffered formalin is used for tissues immunostained withLef-1, Ki67, and hair keratin (AE13) antibodies. Samples are embedded inparaffin and 8 m sections cut for immunostaining. Immunoreactivity ofantigens in formalin-fixed sections is restored by immersing slides in aboiling 0.01 M citrate buffer, pH 6, for 10 minutes. The followingprimary antibodies are used at the indicated dilutions forimmunostaining: rabbit anti-keratins K 1, K 10, K5 and K 14 (1:500)(Roop et al., 1984), loricrin and filaggrin (1:500) (Roop et al., 1987);rabbit anti-K17 (1:1000) (McGowan and Coulombe, 1998); rabbit anti-Lef-1(1:200) (Travis et al., 1991); rabbit anti-Ki67, NCL-Ki67p (NovocastraLaboratories, Ltd., Newcastle upon Tyne, UK) (1:200); and mousemonoclonal AE 13 hybridoma supernatant, which recognize type I hairkeratins (1:5) (Lynch et al., 1986), as described in Beachy et al(2007). Tissue sections are incubated with primary antibodies diluted intris-buffered saline containing 1% bovine serum albumin, typically for1-2 hours at room temperature. Subsequent immunostaining procedures areperformed using peroxidase Vectastain ABC kits (Vector Laboratories,Inc., Burlingame, Calif.) and 3,3′diaminobenzidine (Sigma, St. Louis,Mo.) as a substrate, according to the manufacturers' recommendations.Sections are counterstained with hernatoxylin and mounted using Permount(Fisher Scientific, Pittsburgh, Pa.).

In Situ Hybridization

Non-radioactive RNA in situ hybridization is performed on 5 m sectionsessentially as described (Groves et al., 1995), using previouslydescribed sequences for Gli1 (Walterhouse et al., 1993), Ptc1 (Goodrichet al., 1996), and BMP-4 (Jones et al., 1991).

Vibrissa Follicle Explants

Vibrissa follicle explants are established using CD-I mouse embryos at13.5 days of gestation according to a previously described protocol(Hirai et al., 1989), with minor modifications. Vibrissa pads aretransferred onto Nuclepore filters (13 mm, 8 m pores), and floated on, 2ml of medium [DMEM (Life Technologies, Gaithersburg, Md.)+Ham's F12medium (Life Technologies) (1:1), with 1% FCS (Intergen, Purchase,N.Y.), penicillin (50 units/ml) and streptomycin (50 gg/ml) (LifeTechnologies)] in 6-well plates. Similar results are obtained using aDMEM-based medium, without the addition of Ham's F12. Explants are fedfresh medium every two days. Microdissection is performed with the aidof a Nikon SMZ-2T stereomicroscope and photomicrographs were taken usingan Olympus OM-4 camera. Cyclopamine is stored at −20 as a 10 mM stock in95% EtOH.

RNA Isolation and RT-PCR

RNA is obtained by solubilizing individual explants in TriZol (LifeSciences) and isolating as recommended by the manufacturer. cDNA issynthesized using SuperScript II Rnase H reverse transcriptase withrandom primers (Life Technologies), and RT-PCR performed using theprimers set forth in Beachy et al's, (2007) (Walterhouse et al., 1993).The following PCR conditions are used for MHKA1, Hacl-1, and actin:95.times.3 min “hot start”, 95.times.50 sec, 58.times.30 sec, and72times.60 sec for 25 (actin) or 35 cycles (MHKA 1 and Hacl-1);72.times.7 min. PCR conditions for profilaggrin primers were aspreviously described (Bickenbach et al., 1995). Reaction products arerun through 1.5% agarose gels and visualized with ethidiurn bromide.

Early stages of hair follicle development appear similar in control andShh −/− embryos. Hair germs, consisting of clusters of columnar basalkeratinocytes protruding into the developing dermis with associateddermal condensates, are detected in the skin of both mutant and controlembryos at 15.5 days of gestation. Despite the similar morphology ofcontrol and Shh-deficient hair germs, a dramatic difference in geneexpression patterns is revealed by in situ hybridization. The level ofGli1 mRNA is markedly reduced in both the epithelial and mesenchymalcomponents of Shh −/− primary hair germs. In addition, expression ofPtc1 is reduced in Shh mutant hair germs, although some placodes containlevels slightly above background. These findings are consistent withprevious reports identifying Shh as a positive regulator of both Gli1and Ptc1 (Marigo and Tabin, 1996; Marigo et al., 1996; Lee et al., 1997;Sasaki et al., 1997), and suggest that Shh is signaling in both theepithelial and mesenchymal cells of the developing follicle. In contrastto Gli1 and Ptc1, BMP-4 mRNA is clearly detectable in condensates ofmutant and control embryos, arguing against a requirement for Shh in theinduction of BMP-4 expression. Thus, although Shh is not required forthe initiation of hair follicle development, primary hair germs thatarise in Shh mutant skin are deficient in the expression of at leastsome Shh target genes.

In control embryos, the interval between E15.5 and E17.5 is marked byrapid proliferation and downgrowth of the follicle into the developingdermis, accompanied by a several-fold increase in the mass of thefollicle epithelium and reorganization into distinct cellularcompartments. In the most mature follicles, keratinocytes in the mostperipheral cell layer, which give rise to the outer root sheath in themature follicle, assume a columnar arrangement perpendicular to the longaxis of the developing follicle; cells located centrally are without adefinite orientation at this stage but will eventually be replaced bythe three concentric layers of inner root sheath cells and the threecell types comprising the hair shaft; and the epithelial cells of thedeepest portion of the follicle, the future hair bulb, have surroundedwhat, is at this stage a well-defined cluster of mesenchymal cells, thedermal papilla. Even the less mature follicles exhibit an organized“cap” of mesenchymal cells at their invaginating tips. In strikingcontrast, hair follicles in skin from mutant embryos at E 17.5 fail todevelop past the hair germ stage seen at E 15.5. Although the follicleepithelium is most obviously affected due to its lack of growth,organizing dermal condensates and dermal papillae are conspicuouslyabsent in mutant skin. These results are consistent with the idea thatepidermis-derived Shh (Bitgood and McMahon, 1995; Iseki et al., 1996;Oro et al., 1997; Motoyama et al., 1998) functions as a paracrine signalregulating development of the mesenchymal component of the hairfollicle. Inhibition of follicle formation is not likely to be due to ageneral disruption of skin development since epidermal morphogenesis,marked by the appearance of granular and cornified cell layers, tookplace by E 17.5 in both control and mutant embryos.

Additional studies are performed to determine whether Shh influenced theexpression of epithelial differentiation markers in embryonic skin.Keratinocytes in developing hair follicles can be distinguished by arelative deficiency of K5 and K14, keratins that are abundant insurrounding epidermal basal cells (Kopan and Fuchs, 1989; Byrne et al.,1994). Immunohistochemical staining of E17.5 embryos reveals greatlyreduced or undetectable levels of K14 in a sub-population of cellscomprising the normal follicles in control embryos as well as theprimordial follicles seen in Shh −/− embryos. Moreover, K17, which isnormally not detected in interfollicular epidermis but is expressed indeveloping and mature hair follicles (Panteleyev et al., 1997; McGowanand Coulombe, 1998), is localized to the follicular epithelium in bothcontrol and mutant skin. Thus, although morphogenesis of hair folliclesin Shh −/− skin fails to progress past the hair germ stage, thesestructures contain epithelial cells that have initiated a terminaldifferentiation program characteristic of developing folliclekeratinocytes. Consistent with these morphological findings, theexpression level of epidermal-specific differentiation markers (keratins1 and 10, loricrin, and filaggrin) in Shh −/− skin is similar to orgreater than in control epidermis, based on immunchistochemicalstaining.

Since Shh −/− mice are not viable, post-natal analysis of mutant skin isperformed following grafting onto nude mice. Whereas skin from controlmice produced abundant pigmented hairs, transplanted Shh −/skin failedto generate detectable hairs but exhibited a pigmented graft site,consistent with the strain of donor skin. The histology of control skingrafts reveals the typical structures seen in normal mouse skin,including numerous hair follicles and sebaceous glands. In strikingcontrast, mutant skin failed to produce normal-appearing follicles, hairshafts, or sebaceous glands, but in some cases exhibit a thickenedepidermis with focal areas of hyperkeratosis. Conspicuous aggregates ofbasophilic cells with scant cytoplasm are detected at thedermal-epidermal junction in these mutant grafts. Interestingly, themorphology of cells in the Shh-deficient keratinocyte aggregates isreminiscent of cells in control hair bulbs, and additional analysesrevealed biochemical similarities. Cells in these aggregates areunreactive with K5 antibodies, exhibit abundant nuclear Lef-1 expression(Zhou et al., 1995), and contain a high proportion of proliferatingcells detected by Ki67 immunostaining. Interestingly, short columnarstructures resembling abortive hair shafts are associated with some ofthe Shh mutant keratinocyte aggregates. Moreover, these structuresexpress hair-specific keratin, indicating that an advanced stage in thefollicle differentiation program is achieved despite a dramaticdisruption of normal morphogenesis. Rarely, a small cluster ofmesenchymal cells is seen associated with the base of a keratinocyteaggregate, where these cells immunostain with Lef-1 antibody. Thesefindings suggest that a rudimentary dermal papilla is present in atleast some of the hair germs seen in Shh mutant grafts.

To better define the temporal requirement for Shh during follicledevelopment, tissue culture studies are performed using cyclopamine(GaTield and Keeler, 1996), which has recently been shown to block Shhsignaling in neural plate explants (Cooper et al., 1998; Incardona etal., 1998). Explants are established using vibrissa pads from mice at13.5 days of gestation (Hirai et al., 1989). When grown for six to eightdays in culture, explants undergo robust morphogenesis resulting in theformation of elongated, grossly normal-appearing vibrissa follicles.These follicles contain hair shafts and express genes encoding mousehair keratin A I (MHKA 1) (Kaytes et al., 1991) and a haircortex-specific marker Hacl-1 (Huh et al., 1994), detected by RT-PCR(FIG. 11B). Treatment of explants with cyclopamine results in strikinginhibition of morphogenesis, indicating that Shh signaling is requiredduring or shortly after the hair germ stage of vibrissa, follicledevelopment. In keeping with results using Shh mutant skin,hair-specific transcripts are detected in cyclopamine-treated graftsdespite their altered development, providing further support for thenotion that biochemical differentiation of the follicle is notnecessarily coupled to its morphogenesis. Both control andcyclopamine-treated explants accumulate profilaggrin mRNA, indicatingthat disruption of Shh signaling does not inhibit epidermaldifferentiation.

Fetal Alcohol Syndrome Examples

Example 7

Female rats are administered alcohol for 2 weeks and then mated. Thealcohol administration continues through out pregnancy and the rate offetal alcohol syndrome related defects in the offspring are noted. Asecond set of rats from the same strain are mated without having thealcohol treatment as a control and the rate of defects of the same typeas noted in the first arm are noted. The control arm does not displayany significant number of the defects that are noticed in the alcoholtreatment arm. A third arm of the study includes rats that areadministered D-chiroinositol or a phosphorylate thereof before andduring the alcohol treatment and are otherwise treated in the samemanner as in the alcohol treatment above. The rate of fetal alcoholsyndrome defects noted in the inositol treatment group is substantiallyless than the rate of detects seen in the inositol free alcohol treatedgroup.

Example 8

Example 7 is repeated except that a cholesterol reducing medication isused instead of the alcohol treatment. Similar results to that inExample 1 are obtained.

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All of the references cited above and throughout the specification arehereby incorporated by reference herein.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

In general inositol (underivitzed) can be recovered in the myo form fromplant sources. Chemical synthesis of the underivitized inositolenantiomers is known in the art and many are available, although many insmall quantities not readily convenient for commercial batch production.Pinitol is another known compound which is a monomethyl ester of aninositol isomer. Conversion of these known compounds to the deoxoversions can generally be accomplished by virtue of the Barton Mccombiedeoxygenation reaction, exemplified with respect to nucleosides in U.S.Pat. No. 6,822,089, incorporated herein by reference. Esterification ofone or more of the hydroxy groups of the inositol structure withphosphoric acid, pyrophosphoric acid, carboxylic acids, carbonic acids,sulfonic acid, sulfouous acid and further esters of the other hydroxygroups of these acidic compounds can be achieved by techniques availableto the synthetic chemist. Techniques for the introduction of othersubstituents such as halogens, oxo groups, and substituents having acarbon atom bound to the inositol ring will also be known to those ofordinary skill in the synthetic chemistry art. Phosphorylation may alsobe obtained in a variety of substitution patterns by completephosphorylation of the inositol and then exposure to specific enzymes orbacterial for specific dephosphorylation of particular positions,leaving different phsophoryaltion patterns. Once prepared, the variousphosphorylates can be separated from each other by commonly knownanalytical and separation techniques such as HPLC etc.

Compositions of the invention include those of an inositol, an inositolderivative, an inositol metabolite, polysaccharides containing inositol,and inositol containing phospholipid and an additional active agentwhich is not an inositol. Where the additional active agent is a folate,it is administered in an amount equivalent to an amount of folic acidselected from about 200 μg, about 250 μg, about 300 μg, about 350 μg,about 400 μg, about 450 μg, about 500 μg, about 600 μg, about 650 μg,about 700 μg, about 750 μg, about 800 μg, about 850 μg, about 900 μg,about 950 μg, about 1 mg, about 1.05 mg, about 1.1 mg, about 1.15 mg,about 1.2 mg, about 1.25 mg, about 1.3 mg, about 1.35 mg, about 1.4 mg,about 1.45 mg, about 1.5 mg, about 1.55 mg, and about 1.6 mg per day.Birth defects within the invention uses include those where the defectis VATER/VACTERL association (vertebral [defects], [imperforate] anus,tracheoesophageal [fistula], radial and renal [dysplasia]),rachischisis, (aka spinal dysraphism) such as spina bifida (including,but not limited to spina bifida aperta (aka spinabifida cystica);spinabifida occulta; and occult spinal disorder, among others) and (b)craniorachischisis (aka cranial dysraphism) such as cranium bifida (akaencephalocele or craniocele) each of spina bifida and cranium bifidabeing of any of the following types meningocele, myelomeningocele,lipomeningocele, and lipomyelomeningocele among others; (c) anencephaly;and (d) chiari malformation; (2) caudal regression syndrome, caudaldysplasia sequence, congenitalsacral agenesis; sironmelia (mermaidsyndrome), sacral regression and the like; (3) cranio-facial defectssuch as, without limitation, facial cleft (aka prosopoanoschisis,including without limitation cleft palate, cleft lip, velopharyngealmalformation (including without limitation bifid uvula), etc.); (4)anorectal malformations including, but not limited to (a) imperforateanus, (b) rectoperineal fistula, (c) recto-bladder neck fistula; (d)persistent urogenital sinus, (e) persistent cloaca, etc.; bucket-handlemalformation; among others, acrocallosal syndrome, Basal cell nevussyndrome, bardet-Biedl syndrome, Biemond syndrome,Ectrodactyly-ectoderma dysplasia, cleft lip/palate, Ellis Van Creveldsyndrome, meckel Gruger syndrome McKusick-kaufman syndrome, Mirror handdeformity (ulnar dimelia) Mohr syndrome, oral-facial-digital syndrome,Pallister Hall syndrome, Greig cephalopolysyndactyl), Post axialpolydactyly, GreigRubinstein-Taybi syndrome, Cardiofaciocutaneoussyndrome, noonan syndrome, short rib polydactyly, extra deformed fingersand toes, Lowe syndrome including ocular and renal defects, mentalretardation. This inositol type compound, whether inositol or aninositol derivative, metabolite, polysaccharides containing inositol,and inositol containing phospholipid is administered in an amount thatis equivalent to an amount of D-chiroinositol selected from the groupconsisting of about 0.1 mg, about 1 mg, about 2 mg, about 3 mg, about 4mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10mg, about 15 mg, about 20 mg, about 25 mg, about 50 mg, about 75 mg,about 100 mg, about 125 mg, about 150 mg about 200 mg, about 250 mg,about 300 mg, about 350 mg, about 400 mg, about 500 mg, about 600 mg,about 700 mg, about 750 mg, about 800 mg, about 900 mg, about 950 mg,about 1 g per day, about 1.2 g per day, about 1.8 g per day, about 2 gper day, about 2.5 g per day, about 3 g per day, about 5 g per day,about 10 g per day, about 12 g per day, about 18 g per day, about 24 gper day, about 30 g per day, about 45 g per day, and about 60 g per day.Certain methods include administration to patients where the patient isa female receiving at least one treatment selected from birth control,hormonal replacement therapy, or antiandrogenic therapy; or said patientis a male receiving at least one treatment selected from estrogenictreatment and hormonal ablative therapy; or said patient is a male tofemale trans-sexual receiving at least one therapy selected fromestrogenic treatment and antiandrogenic treatment, more specifically insome patients, the estrogenic insult is from a medicinal source and saidmedicinal source is selected from the group consisting of estrogenic,progestogenic, or antiandrogenic therapy. In some embodiments, thetreatment is of a cancer selected from basal cell carcinoma (shh gain offunction), Multiple basal cell nevi, squamous cell carcinoma (ptcactivity) medulloblastoma, primitive neuroectodermal tumor (PNET),Gorlin syndrome, nevoid basal cell carcinoma syndrome, harmartomas, bluerubber-bleb nevus syndrome, Turcot syndrome, glioma polyposis syndrome,Rubinstein-Taybi syndrome, Cowden tumor syndrome, rabdomyosarcoma (RMS),alveolar rhabdomyosarcoma, botryoid rhabdomyosarcoma, embryonalrhabdomyosarcoma, spindle cell rhabdomyosarcoma, pleomorphicrhabdomyosarcoma, soft tissue sarcoma, rhabdomyoblasts, pediatricsarcoma, cell carcinoma, carcinosarcoma, adenocystic carcinoma,epidermoid carcinoma, nasopharyngeal carcinoma, bladder carcinoma, renalcell carcinoma, papilloma, or an epidermoidoma. In other embodiments,the birth defect is VATER/VACTERL association (vertebral [defects],[imperforate] anus, trachcoesophageal [fistula], radial and renal[dysplasia])rachischisis (aka spinal dysraphism) such as spina bifida(including, but not limited to spina bifida aperta (aka spinabifidacystica); spinabifida occulta; and occult spinal disorder, among others)and (b) craniorachischisis (aka cranial dysraphism) such as craniumbifida (aka encephalocele or craniocele) each of spina bifida andcranium bifida being of any of the following types meningocele,myelomeningocele, lipomeningocele, and lipomyelomeningocele amongothers; (c) anencephaly; and (d) chiari malformation; (2) caudalregression syndrome, caudal dysplasia sequence, congenitalsacralagenesis; sironmelia (mermaid syndrome), sacral regression and the like;(3) cranio-facial defects such as, without limitation, facial cleft (akaprosopoanoschisis, including without limitation cleft palate, cleft lip,velopharyngeal malformation (including without limitation bifid uvula),etc.); (4) anorectal malformations including, but not limited to (a)imperforate anus, (b) rectoperineal fistula, (c) recto-bladder neckfistula; (d) persistent urogenital sinus, (e) persistent cloaca, etc.;(5) bucket-handle malformation; among others. Biemond syndrome,Ectrodactyly-ectoderma dysplasia, cleft lip/palate, Ellis Van Creveldsyndrome, Muir-Torre syndrome, Cowden syndrome, Carney complex,Birt-Hogg-Dube syndrome, Gorlin syndrome (ptc loss-of-function),Gorlin-Goltz syndrome, basal cell nevus syndrome, bifid-rib basal-cellnevus syndrome, multiple basal cell nevi, Meckel Gruger syndrome,McKusick-Kaufmansyndrome, Mirror hand deformity (ulnar dimelia) Mohrsyndrome, Oral-facial-digital syndrome, Pallister Hall syndrome,cephalopolysyndactyl), Post axial polydactyly, GreigRubinstein-Taybisyndrome, retinoblastoma, Cardiofaciocutaneous syndrome, Noonansyndrome, short rib polydactyly, extra deformed fingers and toes, Lowesyndrome (including ocular and renal defects), Renal Colombo syndrome,Retinitis pigmentosa. In some embodiments cotherapy of an inositol orderivative thereof or metabolite thereof or of a polysaccharide havinginositol components or an inositol phospholipid is administered as acotherwith other active agents to a patient selected from the groupselected from (a) those in need of preventing of inhibiting ofproliferation, growth, and/or metastases of tissues or conditionsselected from the group consisting of breast tissue, prostate tissue,cervical cancer caused by human papiloma virus subtypes (HPV), Kaposissarcoma, lung cancer, adenocarcinoma, gut derived tumors, colon cancersdue to adenocarcinomas, and human erythroleukemia and (b) those in needof tissue regulation selected from the group consisting of regulation ofneural tissues, regulation of bone and cartilage formation and repair,regulation of ovulation, regulation of spermatogenesis, regulation ofsmooth muscle, regulation of lung, liver, intestines, colon, rectum andother organs arising from the primitive gut as well as the distalhindgut, regulation of hematopoietic function, hemopoietic stein cells,and regulation of skin and hair growth, and modulation of cellproliferation which includes, without limitation, inhibition ofangiogenesis. The modulation of cell proliferation also includes a basalcell carcinoma, medulloblastoma, primitive neuroectodermal tumor, PNET,Gorlin syndrome, nevoid basal cell carcinoma syndrome, harmartomas, bluerubber-bleb nevus syndrome, Turcot syndrome, glioma polyposis syndrome,Rubinstein-Taybi syndrome, Cowden tumor syndrome, rabdomyosarcoma, RMS,alveolar rhabdomyosarcoma, botryoid rhabdomyosarcoma, embryonalrhabdomyosarcoma, spindle cell rhabdomyosarcoma, pleomorphicrhabdomyosarcoma, soft tissue sarcoma, rhabdomyoblasts, pediatricsarcoma, sarcoma squamous cell carcinoma, carcinosarcoma, adenocysticcarcinoma, epidermoid carcinoma, nasopharyngeal carcinoma, bladdercarcinoma, renal cell carcinoma, papilloma, karposi's sarcoma, and anepidermoidoma. The inhibition of cell proliferation also includespreventing or inhibiting the proliferation, growth, and/or metastases ofone or more cancers selected from the group consisting of breast cancer,prostate cancer, especially prostatic androgen dependent PCA-LNA-pcells, cervical cancer, caused by human papiloma virus subtypes (HPV),Kaposis sarcoma, lung cancer, (in particular, small cell lung cancer,adenocarcinoma; gut derived tumors (including but not limited to cancerof the esophagus, stomach, pancreas, biliary duct system, intestinal(gastric) system, colon cancers due to adenocarcinomas, and humanerythroleukemia.

The instant compounds may also be co-administered with other well knowntherapeutic agents that are selected for their particular usefulnessagainst the condition that is being treated.

For example, instant compounds are useful in combination with knownanti-cancer agents. Combinations of the presently disclosed compoundswith other anti-cancer or chemotherapeutic agents are within the scopeof the invention. Examples of such agents can be found in CancerPrinciples and Practice of Oncology by V. T. Devita and S. Hellman(editors), 8th edition (April, 2008), Lippincott Williams & WilkinsPublishers. A person of ordinary skill in the art would be able todiscern which combinations of agents would be useful based on theparticular characteristics of the drugs and the cancer involved. Suchanti-cancer agents include the following: estrogen receptor modulators,androgen receptor modulators, retinoid receptor modulators,cytotoxic/cytostatic agents, antiproliferative agents, prenyl-proteintransferase inhibitors, HMG-CoA reductase inhibitors and otherangiogenesis inhibitors and agents that interfere with cell cyclecheckpoints. The instant compounds are particularly useful whenco-administered if needed with radiation therapy.

“Estrogen receptor modulators” refers to compounds that interfere withor inhibit the binding of estrogen to the receptor, regardless ofmechanism. Examples of estrogen receptor modulators include, but are notlimited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081,toremifene, fulvestrant,4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]ph-enyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

“Androgen receptor modulators” refers to compounds which interfere orinhibit the binding of androgens to the receptor, regardless ofmechanism. Examples of androgen receptor modulators include finasterideand other 5.alpha-reductaseinhibitors, nilutamide, flutamide,bicalutamide, liarozole, and abiraoacetate.

“Retinoid receptor modulators” refers to compounds which interfere orinhibit the binding of retinoids to the receptor, regardless ofmechanism. Examples of such retinoid receptor modulators includebexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid,alpha-difluoromethylomithine, ILX23-7553,trans-N-(4′-hydroxyphenyl)retinamide, N-4-carboxyphenyl, retinamide.

“Cytotoxic/cytostatic agents” refer to compounds which cause cell deathor inhibit cell proliferation primarily by interfering directly with thecell's functioning or inhibit or interfere with cell myosis, includingalkylating agents, tumor necrosis factors, intercalators, hypoxiaactivatable compounds, microtubule inhibitors/microtubule-stabilizingagents, inhibitors of mitotic kinesins, anti-metabolites; biologicalresponse modifiers; hormonal/anti-hormonal therapeutic agents,haematopoietic growth factors, monoclonal antibody targeted therapeuticagents and topoisomerase inhibitors.

Examples of cytotoxic agents include, but are not limited to, sertenef,cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine,prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin,oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfantosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa,lobaplatin, satraplatin, profiromycin, cisplatin, irofulven,dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum,benzylguanine, glufosfamide, GPX100,(trans,trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]-bis[diamine(chloro)platinum(II)]tetrachloride,diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycaminomycin, annamycin,galarubicin, elinafide, MEN10755, and4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (seeWO00/50032). An example of a hypoxia activatable compound for cancer istirapazmine.

Examples of microtubule inhibitors/microtubule-stabilizing agentsinclude paclitaxel, vindesine sulfate,3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol, rhizoxin,dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881,BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butyla-mide,TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and6,288,237, and BMS188797.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine,iminotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chatreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione,lurtotecan,7-[2-(N-isopropylaminoamino)ethyl]-(20S) camptothecin,BNP1350,BNPI1100,BN80915, BN80942, etoposidephosphate, teniposide, sobuzoxane,2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazo-le-1-carboxamide,asulacrine,(5a,5aB,8aa9b)-9-[2-[N-[2-dimethylamino)ethyl]-N-methylamino]ethyl]-5-[-4-hydroxy-3,5-dimethoxyphenyl]-5,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridiniu-m,6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethy-1]formamide,N-(2-(dimethylamino)ethyl)acridine-4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-on-e,and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the humanmitotic kinesin KSP, are described in PCT Publications WO 01/30768 andWO 01/98278, and pending U.S. Ser. Nos. 60/338,779 (filed Dec. 6, 2001),60/338,344 (filed Dec. 6, 2001), 60/338,383 (filed Dec. 6, 2001),60/338,380 (filed Dec. 6, 2001), 60/338,379 (filed Dec. 6, 2001) and60/344,453 (filed Nov. 7, 2001).

“Antiproliferative agents” includes antisense RNA and DNAoligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001,and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin,doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine,cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed,nelzarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxycytidine,N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea,N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamicacid, aminopterin, 5-fluorouracil, alanosine,11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetr-acyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N-4-palmitoyl-1-B-D-arabino furanosylcytosine,3-aminopyridine-2-carboxaldehydethiosemicarbazone and trastuzamab.

Examples of monoclonal antibody targeted therapeutic agents includethose therapeutic agents which have cytotoxic agents or radioisotopesattached to a cancer cell specific or target cell specific monoclonalantibody. One particular example is Bexxar.

“HMG-CoA reductase inhibitors” refers to inhibitors of3-hydroxy-3-methylglutaryl-CoA reductase. Compounds which haveinhibitory activity for HMG-CoA reductase can be readily identified byusing assays well-known in the art. For example, see the assaysdescribed or cited in U.S. Pat. No. 4,231,938 at col. 6, and WO 84/02131at pp. 30-33. The terms “HMG-CoA reductase inhibitor” and “inhibitor ofHMG-CoA reductase” have the same meaning when used herein.

Examples of HMG-CoA reductase inhibitors that may be used include butare not limited to lovastatin (MEVACOR®; see U.S. Pat. Nos. 4,231,938,4,294,926 and 4,319,039), simvastatin (ZOCOR®; see U.S. Pat. Nos.4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL®; see U.S.Pat. Nos. 4,346,227, 4,537,859, 4,410,629, 5,030,447 and 5,180,589),fluvastatin (LESCOL®; see U.S. Pat. Nos. 5,354,772, 4,911,165,4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896), atorvastatin(LIPITOR®; see U.S. Pat. Nos. 5,273,995, 4,681,893, 5,489,691 and5,342,952) and cerivastatin (also known as rivastatin and BAYCHOL®; seeU.S. Pat. No. 5,177,080). The structural formulas of these andadditional HMG-CoA reductase inhibitors that may be used in the instantmethods are described at page 87 of M. Yalpani, “Cholesterol LoweringDrugs”, Chemistry & Industry, pp. 85-89 (5 Feb. 1996) and U.S. Pat. Nos.4,782,084 and 4,885,314. The term HMG-COA reductase inhibitor as usedherein includes all pharmaceutically acceptable lactone and open-acidforms (i.e., where the lactone ring is opened to form the free acid) aswell as salt and ester forms of compounds which have HMG-CoA reductaseinhibitory activity, and therefor the use of such salts, esters,open-acid and lactone forms is included within the scope of thisinvention.

In HMG-CoA reductase inhibitors where an open-acid form can exist, saltand ester forms may preferably be formed from the open-acid, and allsuch forms are included within the meaning of the term “HMG-CoAreductase inhibitor” as used herein. Preferably, the HMG-CoA reductaseinhibitor is selected from lovastatin and simvastatin, and mostpreferably simvastatin. Herein, the term “pharmaceutically acceptablesalts” with respect to the HMG-CoA reductase inhibitor shall meannon-toxic salts of the compounds employed in this invention which aregenerally prepared by reacting the free acid with a suitable organic orinorganic base, particularly those formed from cations such as sodium,potassium, aluminum, calcium, lithium, magnesium, zinc andtetramethylammonium, as well as those salts formed from amines such asammonia, ethylenediamine, N-methylglucamine, lysine, arginine,ornithine, choline, N,N′-dibenzylethylenediamine, chloroprocaine,diethanolamine, procaine, N-benzylphenethylamine,1-p-chlorobenzyl-2-pyrrolidine1′-yl-methylbenzimidazole, diethylamine,piperazine, and tris(hydroxymethyl)aminomethane. Further examples ofsalt forms of HMG-CoA reductase inhibitors may include, but are notlimited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate,bitartrate, borate, bromide, calcium edetate, camsylate, carbonate,chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate,estolate, esylate, fumarate, gluceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynapthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, maleate, mandelate, mesylate,methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamaote,palmitate, panthothenate, phosphate/diphosphate, polygalacturonate,salicylate, stearate, subacetate, succinate, tannate, tartrate,teoclate, tosylate, triethiodide, and valerate.

Ester derivatives of the described HMG-CoA reductase inhibitor compoundsmay act as prodrugs which, when absorbed into the bloodstream of awarm-blooded animal, may cleave in such a manner as to release the drugform and permit the drug to afford improved therapeutic efficacy.

“Prenyl-protein transferase inhibitor” refers to a compound whichinhibits any one or any combination of the prenyl-protein transferaseenzymes, including farnesyl-protein transferase (FPTase),geranylgeranyl-protein transferase type I (GGPTase-I), andgeranylgeranyl-protein transferase type-II (GGPTase-II, also, called RabGGPTase). Examples of prenyl-protein transferase inhibiting compoundsinclude(.+−.)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,(−)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,(+)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,5(S)-n-butyl-1-(2,3-dimethylphenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmeth-yl]-2-iperazinone,(S)-1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-5-[2-ethanesulfonyl)methyl)-2-piperazinone,5(S)-n-Butyl-1-(2-methylphenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone,1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-2-methyl-5-midazolylmethyl]-2-piperazinone,1-(2,2-diphenylethyl)-3-[N-(1-(4-cyanobenzyl)-1H-imidazol-5-ylethyl)carba-moyl]piperidine,4-{5-[4-hydroxymethyl-4-(4-chloropyridin-2-ylmethyl)-piperidine-1-ylmethy-1]-2-methylimidazol-1-ylmethyl}benzonitrile,4-{5-[4-hydroxymethyl-4-(3-chlorobenzyl)-piperidine-1-ylmethyl]-2-methyli-midazol-1-ylmethyl}benzonitrile,4-{3-[4-(2-oxo-2H-pyridin-1-yl)benzyl]-3H-imidazol-4-ylmethyl}benzonitril-e,4-{3-[4-(5-chloro-2-oxo-2H-[1,2′]bipyridin-5′-ylmethyl]-3H-imidazol-4-y-lmethyl}benzonitrile,4-{3-[4-(2-oxo-2H-[1,2′]bipyridin-5′-ylmethyl]-3H-imidazol-4-ylmethyl}ben-zonitrile,4-{3-(2-oxo-1-phenyl-1,2-dihydropyridin-4-ylmethyl)-3H-imidazol-4-ylmethyl}benzonitrile,18,19-dihydro-19-oxo-5H,17H-6,10:12,16-dimetheno-1H-imidazo[4,3-c][1,11,4]-dioxaazacyclononadecine-9-carbonitrile,(.+−.)-19,20-dihydro-19-oxo-5H-18,21-ethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxatriaza-cyclooctadecine-9-carbonitrile,19,20-dihydro-19-oxo-5H,17H-18,21-ethano-6,10:12,16-dimetheno-22H-imidazo-[3,4-h][1,8,11,14]oxatriaxacyclocicosine-9-carbonitrile,and(+)-19,20-dihydro-3-methyl-19-oxo-5H-18,21-ethano-12,14-etheno-6,10-methe-no-22H-benzo[d-]imidazo[4,3-k][1,6,9,12]oxa-triazacyclcooctadecine-9-carbonitrile.

Other examples of prenyl-protein transferase inhibitors can be found inthe following publications and patents: WO 96/30343, WO 97/18813, WO97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO95/32987, U.S. Pat. Nos. 5,420,245, 5,523,430, 5,532,359, 5,510,510,5,589,485, 5,602,098, European Patent Publ. 0 618 221, European PatentPubl. 0 675 112, European Patent Publ. 0 604 181, European Patent Publ.0 696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO 95110515, WO95/10516, WO 95/24612, WO 95134535, WO 95/25086, WO 96/05529, WO96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO 96/33159, WO96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO98/02436, and U.S. Pat. No. 5,532,359. For an example of the role of aprenyl-protein transferase inhibitor on angiogenesis see European J. ofCancer, Vol. 35, No. 9, pp. 1394-1401 (1999).

“Angiogenesis inhibitors” refers to compounds that inhibit the formationof new blood vessels, regardless of mechanism. Examples of angiogenesisinhibitors include, but are not limited to, tyrosine kinase inhibitors,such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) andFlk-1/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived,or platelet derived growth factors, MMP (matrix metalloprotease)inhibitors, integrin blockers, interferon-α, interleukin-12, pentosanpolysulfate, cyclooxygenase inhibitors, including nonsteroidalanti-inflammatories (NSAIDs) like aspirin and ibuprofen as well asselective cyclooxygenase-2 inhibitors like celecoxib and rofecoxib(PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch.Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec., Vol. 238, p. 68 (1994);FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol. 313, p. 76(1995); J. Mol. Endocrinol., Vol. 16, p. 107 (1996); Jpn. J. Pharmacol.,Vol. 75, p. 105 (1997); Cancer Res., Vol. 57, p. 1625 (1997); Cell, Vol.93, p. 705 (1998); Intl. J. Mol. Med., Vol. 2, p. 715 (1998); J. Biol.Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such ascorticosteroids, mineralocorticoids, dexamethasone, prednisone,prednisolone, methylpred, betamethasone), carboxyamidotriazole,combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol,thalidomide, angiostatin, troponin-1, angiotensin 11 antagonists (seeFernandez et al., J. Lab. Clin. Med. 105:141-145 (1985)), and antibodiesto VEGF (see, Nature Biotechnology, Vol. 17, pp. 963-968 (October 1999);Kim et al., Nature, 362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Other therapeutic agents that modulate or inhibit angiogenesis and mayalso be used in combination with the compounds of the instant inventioninclude agents that modulate or inhibit the coagulation and fibrinolysissystems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examplesof such agents that modulate or inhibit the coagulation and fibrinolysispathways include, but are not limited to, heparin (see Thromb. Haemost.80:10-23 (1998)), low molecular weight heparins and carboxypeptidase Uinhibitors (also known as inhibitors of active thrombin activatablefibrinolysis inhibitor [TAFIa] (see Thrombosis Res. 101:329-354 (2001)).TAFIa inhibitors have been described in U.S. Ser. Nos. 60/310,927 (filedAug. 8, 2001) and 60/349,925 (filed Jan. 18, 2002).

“Agents that interfere with cell cycle checkpoints” refer to compoundsthat inhibit protein kinases that transduce cell cycle checkpointsignals, thereby sensitizing the cancer cell to DNA damaging agents.Such agents include inhibitors of ATR, ATM, the Chk1 and Chk2 kinasesand cdk and cde kinase inhibitors and are specifically exemplified by7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

As described above, the combinations with NSAID's are directed to theuse of NSAID's which are potent COX-2 inhibiting agents. For purposes ofthis specification an NSAID is potent if it possesses an IC₅₀ for theinhibition of COX-2 of 1 mu.M or less as measured by cell or microsomalassays.

The invention also encompasses combinations with NSAID's which areselective COX-2 inhibitors. For purposes of this specification NSAID'swhich are selective inhibitors of COX-2 are defined as those whichpossess a specificity for inhibiting COX-2 over COX-1 of at least 100fold as measured by the ratio of IC₅₀ for COX-2 over IC₅₀ for COX-1evaluated by cell or microsomal assays. Such compounds include, but arenot limited to those disclosed in U.S. Pat. No. 5,474,995, issued Dec.12, 1995, U.S. Pat. No. 5,861,419, issued Jan. 19, 1999, U.S. Pat. No.6,001,843, issued Dec. 14, 1999, U.S. Pat. No. 6,020,343, issued Feb. 1,2000, U.S. Pat. No. 5,409,944, issued Apr. 25, 1995, U.S. Pat. No.5,436,265, issued Jul. 25, 1995, U.S. Pat. No. 5,536,752, issued Jul.16, 1996, U.S. Pat. No. 5,550,142, issued Aug. 27, 1996, U.S. Pat. No.5,604,260, issued Feb. 18, 1997, U.S. Pat. No. 5,698,584, issued Dec.16, 1997, U.S. Pat. No. 5,710,140, issued Jan. 20, 1998, WO 94/15932,published Jul. 21, 1994, U.S. Pat. No. 5,344,991, issued Jun. 6, 1994,U.S. Pat. No. 5,134,142, issued Jul. 28, 1992, U.S. Pat. No. 5,380,738,issued Jan. 10, 1995, U.S. Pat. No. 5,393,790, issued Feb. 20, 1995,U.S. Pat. No. 5,466,823, issued Nov. 14, 1995, U.S. Pat. No. 5,633,272,issued May 27, 1997, and U.S. Pat. No. 5,932,598, issued Aug. 3, 1999,all of which are hereby incorporated by reference.

General and specific synthetic procedures for the preparation of theCOX-2 inhibitor compounds described above are found in U.S. Pat. No.5,474,995, issued Dec. 12, 1995, U.S. Pat. No. 5,861,419, issued Jan.19, 1999, and U.S. Pat. No. 6,001,843, issued Dec. 14, 1999, all ofwhich are herein incorporated by reference.

Other examples of angiogenesis inhibitors include, but are not limitedto, endostatin, ukrain, ranpirnase, IM862,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino-1-[[3,5-ichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triaz—ole-4-carboxamide,CM101,qualamine, combretastatin, RP14610, NX31838, sulfated mannopentaosephosphate,7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-py-rrole]-carbonylimino]-bis-(1,3-naphthalenedisulfonate),and 3-[(2,4-dimethylpyrrol-5-ylmethylene]-2indolinone (SU5416).

As used above, “integrin blockers” refers to compounds which selectivelyantagonize, inhibit or counteract binding of a physiological ligand tothe .alpha.sub.v.beta.sub.3 integrin, to compounds which selectivelyantagonize, inhibit or counter-act binding of a physiological ligand tothe alpha.v.beta.5 integrin, to compounds which antagonize, inhibit orcounteract binding of a physiological ligand to both thealpha.sub.v.beta.sub.3 integrin and the .alpha.sub.v.beta.sub 5integrin, and to compounds which antagonize, inhibit or counteract theactivity of the particular integrin(s) expressed on capillaryendothelial cells. The term also refers to antagonists of thealpha.sub.v.beta.sub.6.alspha.su.v.beta.sub.8,alpha.sub.1.beta.sub.1,alpha.sub.2.beta.sub.1.alpha.sub.5.beta.sub.1, alpha.sub.6.beta.sub.1and alpha.sub.6.beta.sub.4 integrins. The term also refers toantogonists of any combination ofalpha.sub.v.beta.sub.3,alpha.sub.v.beta.sub.5.alpha.sub.v.beta.sub.6alpha.sub.v.beta.sub.8,alpha.sub.1beta.sub.1,alpha.sub.2.beta.sub.1,alpha.sub.5.beta.sub.1, alpha.sub.6.beta.sub.1 andalpha.sub.6.beta.sub.4 integrins.

Some specific examples of tyrosine kinase inhibitors includeN-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]q-uinazoline,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,BIBX1382,2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epox-y-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazo-cin-1-one,SH268, genistein, ST1571, CEP2563,4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethanesulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, ST1571A,N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD 121974.

Combinations with compounds other than anti-cancer compounds are alsoencompassed in the instant methods. For example, combinations of theinstantly claimed compounds with PPAR-.gamma (i.e., PPAR-gamma) agonistsand PPAR-delta. (i.e., PPAR-delta) agonists are useful in the treatmentof certain malingnancies. PPAR-gamma and PPAR-delta are the nuclearperoxisome proliferator-activated receptors gamma and delta. Theexpression of PPAR-gamma on endothelial cells and its involvement inangiogenesis has been reported in the literature (see J. Cardiovasc.Pharmacol. 1998; 31:909-913; J. Biol. Chem. 1999; 274:9116-9121; Invest.Ophthalmol Vis. Sci. 2000; 41:2309-2317). More recently, PPAR.gammaagonists have been shown to inhibit the angiogenic response to VEGF invitro; both troglitazone and rosiglitazone maleate inhibit thedevelopment of retinal neovascularization in mice. (Arch. Ophthamol.2001; 119:709-717). Examples of PPAR-gamma agonists and PPAR-gamma/alphaagonists include, but are not limited to, thiazolidinediones (such asDRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone),fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR-H-1039242,JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158,NN622, G1262570, PNU182716, DRF552926,2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpro-pionicacid (disclosed in U.S. Ser. No. 09/782,856), and2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2--carboxylic acid (disclosed in U.S. Ser. No. 60/235,708 and 60/244,697).

Another embodiment of the instant invention is the use of the presentlydisclosed compounds in combination with gene therapy for the treatmentof cancer. For an overview of genetic strategies to treating cancer seeHall et al (Am J Hum Genet 61:785-789, 1997) and Kufe et al (CancerMedicine, 5th Ed, pp 876-889, B C Decker, Hamilton 2000). Gene therapycan be used to deliver any tumor suppressing gene. Examples of suchgenes include, but are not limited to, p53, which can be delivered viarecombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134,for example), a uPA/uPAR antagonist (“Adenovirus-Mediated Delivery of auPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth andDissemination in Mice,” Gene Therapy, Aug. 5, 1998; (8):1105-13), andinterferon gamma (J Immunol 2000; 164:217-222).

The compounds of the instant invention may also be administered incombination with an inhibitor of inherent multidrug resistance (MDR), inparticular MDR associated with high levels of expression of transporterproteins. Such MDR inhibitors include inhibitors of p-glycoprotein(P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833(valspodar).

A compound of the present invention may be employed in conjunction withanti-emetic agents to treat nausea or emesis, including acute, delayed,late-phase, and anticipatory emesis, which may result from the use of acompound of the present invention, alone or with radiation therapy. Forthe prevention or treatment of emesis, a compound of the presentinvention may be used in conjunction with other anti-emetic agents,especially neurokinin-1 receptor antagonists, 5HT3 receptor antagonists,such as ondansetron, granisetron, tropisetron, and zatisetron, GABABreceptor agonists, such as baclofen, a corticosteroid such as Decadron(dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten orothers such as disclosed in U.S. Pat. Nos. 2,789,118, 2,990,401,3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, anantidopaminergic, such as the phenothiazines (for exampleprochlorperazine, fluphenazine, thioridazine and mesoridazine),metoclopramide or dronabinol. For the treatment or prevention of emesisthat may result upon administration of the instant compounds,conjunctive therapy with an anti-emesis agent selected from aneurokinin-1 receptor antagonist, a 5HT3 receptor antagonist and acorticosteroid is preferred.

Neurokinin-1 receptor antagonists of use in conjunction with thecompounds of the present invention are fully described, for example, inU.S. Pat. Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595,5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719,147; European PatentPublication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 429 366, 0 430771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0512 902, 0 514 273, 0 514 274, 0 514 275, 0 514 276, 0 515 681,0 517589, 0 520 555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0545 478, 0 558 156, 0 577 394, 0 585 913, 0 590 152, 0 599 538, 0 610793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0707 006, 0 708 101, 0 709 375, 0 709 376, 0 714 891,0 723 959, 0 733 632and 0 776 893; PCT International Patent Publication Nos. WO 90/05525,90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151, 92/15585,92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330, 93/00331,93/01159, 93/01165, 93/01169, 93/01170, 93/06099, 93/09116, 93/10073,93/14084, 93/14113, 93/18023, 93/19064, 93/21155, 93/21181, 93/23380,93/24465, 94100440, 94/01402, 94/02461, 94/02595, 94/03429, 94/03445,94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165, 94/10167,94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767, 94/15903,94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309, 95/02595,95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549, 95/11880,95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129, 95/19344,95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418, 95/30674,95/30687, 95/33744, 96/05181, 96105193, 96/05203, 96/06094, 96/07649,96/10562, 96/16939, 96/18643, 96/20197, 96/21661, 96/29304, 96/29317,96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 97/01553, 97/01554,97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084, 97/19942 and97/21702; and in British Patent Publication Nos. 2 266 529, 2 268 931, 2269 170, 2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293 169, and 2302 689. The preparation of such compounds is fully described in theaforementioned patents and publications, which are incorporated hereinby reference.

A neurokinin-1 receptor antagonist for use in conjunction with thecompounds of the present invention is2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluoropheny-1)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)morpholineor a pharmaceutically acceptable salt which is described in U.S. Pat.No. 5,719,147.

A compound of the instant invention may also be administered with anagent useful in the treatment of anemia. Such an anemia treatment agentis, for example, a continuous erythropoiesis receptor activator, such asapoetin alfa.

A compound of the instant invention may also be administered with anagent useful in the treatment of neutropenia. Such a neutropeniatreatment agent is, for example, a hematopoietic growth factor whichregulates the production and function of neutrophils such as a humangranulocyte colony stimulating factor, (G-CSF). Examples of a G-CSFinclude filgrastin.

A compound of the instant invention may also be administered with animmunologic-enhancing drug such as levamisole, Isoprinosine, andzadaxin.

Thus, the scope of the instant invention encompasses the use of theinstantly claimed compounds in combination with a second compoundselected from: 1) PI3k inhibitor, 2) Akt inhibitor, 3) TNF inhibitorsuch as Intlixamab, Humira, Etanercept 4) an estrogen receptormodulator, 5) an androgen receptor modulator, 6) retinoid receptormodulator, 7) a cytotoxic agent, 8) an antiproliferative agent, 9) aprenyl-protein transferase inhibitor, 10) an HMG-CoA reductaseinhibitor, 11) an HIV protease inhibitor, 12) a reverse transcriptaseinhibitor, 13) an angiogenesis inhibitor, 14) an inhibitor of inherentmultidrug resistance, 15) an anti-emetic agent, 16) an agent useful inthe treatment of anemia, 17) agent useful in the treatment ofneutropenia, and 18) an immunologic-enhancing drug in certain cases.

Also included in the scope of the claims is a method of treating cancerthat comprises administering a therapeutically effective amount of oneor more inositol compounds of the invention or derivative thereofdescribed above in combination with radiation therapy and/or incombination with a compound selected from: 1) PI3k inhibitor, 2) Aktinhibitor, 3) TNF inhibitor such as Inflixamab, Humira, Etanercept 4) anestrogen receptor modulator, 5) an androgen receptor modulator, 6)retinoid receptor modulator, 7) a cytotoxic agent, 8) anantiproliferative agent, 9) a prenyl-protein transferase inhibitor, 10)an HMG-CoA reductase inhibitor, 11) an HIV protease inhibitor, 12) areverse transcriptase inhibitor, 13) an angiogenesis inhibitor, 14) aninhibitor of inherent multidrug resistance, 15) an anti-emetic agent,16) an agent useful in the treatment of anemia, 17) agent useful in thetreatment of neutropenia, and 18) an immunologic-enhancing drug.

When a composition according to this invention is administered into ahuman subject, the daily dosage will normally be determined by theprescribing physician with the dosage generally varying according to theage, weight, and response of the individual patient, as well as theseverity of the patients symptom's.

In one exemplary application, in addition to amounts set forth above, asuitable amount of an inhibitor of Akt/PKB is administered to a mammalundergoing treatment for cancer. Administration occurs in an amount ofinhibitor of between about 0.1 mg/kg of body weight to about 60 mg/kg ofbody weight per day, preferably of between 0.5 mg/kg of body weight toabout 40 mg/kg of body weight per day. A particular therapeutic dosagethat comprises the instant composition includes from about 0.01 mg toabout 1000 mg of inhibitor of Akt/PKB. Preferably, the dosage comprisesfrom about 1 mg to about 1000 mg of inhibitor of Akt/PKB.

In addition to the above, the compounds of the invention can be used incombinations with monoclonal antibodies for autoimmune conditiontreatments such as Adalimumab and certolizumab. Still further thecompounds of the invention can be used in combinations with variousactive agents currently in use for chronic, active hepatitis, inparticular, without limitation, pegylated interferon, Ribavarin(copegus, reberol), rebetron, Inteferons, epivir-HBV, inteferonalpha/alpha 2 plus ribavarin combination.

All patents, publications and pending patent applications identified arehereby incorporated by reference.

The compounds of the invention can be also be used in the treatment ofor as a synergistic inhibitor of autoimmune diseases mediated bydefective or overactive signaling pathways: More particularly, thecompounds of the invention can be used in treating patients withAchlorhydra Autoimmune Active Chronic Hepatitis, Addison's Disease,Alopecia, Areata, Amyotrophic Lateral Sclerosis (ALS, Lou Gehrig'sDisease), Ankylosing Spondylitis, Anti-GBM Nephritis or anti-TBMNephritis, Antiphospholipid Syndrome. Aplastic Anemia, RheumatoidArthritis, Asthma, Atopic Allergy, Atopic Dermatitis, Autoimmune InnerEar Disease (AIED), Autoimmune Lymphoproliferative Syndrome (ALPS), BaloDisease, Behcet's Disease, Berger's Disease, (IgA Nephropathy), BullousPemphigoid, cardiomyopathy, Celiac Disease, Chronic Fatigue ImmuneDysfunction Syndrome (CFIDS), Churg Strauss Syndrome, CicatricialPemphigoid, Cogan's Syndrome, Cold Agglutunin Disease, Colitis, CranialArteritis, CREST Syndrome, Crohn's Disease, Cushing's Syndrome, Dego'sDisease, Dermatitis, Dermatomyositis, Devic Disease, Type 1 Diabetes,Type 2 Diabetes, Dressler's Syndrome, Discoid Lupus, Eczema, EssentialMixed cryoglobulinemia, Eosinophilic, Fasciitis, Epidermolysis BullosaAcquisita, Evan's Syndrome, Fibromyalgia, Fibromyositis, FibrosingAlveolitis, Gastritis, Giant Cell Artertis, Glomerulonephritis,Goodpasture's Disease, Grave's Disease, Guillian-Barre Syndrome,Hashimoto's Thyroiditis, Hemolytic Anemia, Henoch-Schonlein Purpura,Hepatitis, Hughes Syndrome, Idiopathic Adrenal Atrophy, IdiopathicPulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura, InflammatoryDemylinating Polyneuropathy, Irritable Bowel Syndrome, Kawasaki'sDisease, Lichen Planus, Lou Gehrig's Disease, Lupoid Hepatitis, Lupus,Lyme Disease, Meniere's Disease, Mixed Connective Tissue Disease,Multiple Myeloma, Multiple Sclerosis, Myasthenia Gravis, Myositis,Ocular Cicatricial Pemphigoid, Osteoporosis, Pars Planitis, PemphigusVulgaris, Polyglandular Autoimmune Syndromes, Polymyalgia Rheumatica(PMR), Polymyositis, Primary Biliary Cirrhosis, Primary SclerosingCholangitis, Psoriasis, Raynaud's Phenomenon, Reiter's Syndrome,Rheumatic Fever, Rheumatoid Arthritis, Sarcoidosis, Scleritis,Scleroderma, Sjogren's Syndrome, Sticky Blood Syndrome, Still's Disease,Stiff Man Syndrome, Sydenham Chorea, Systemic Lupus Erythmatosis (SLE),Takayasu's Arteritis, Temporal Arteritis, Ulcerative Colitis,Vasculitis, Vitiligo, Wegener's Granulomatosis, and Wilson's Syndrome.

The invention claimed is:
 1. A method for treating breast cancer andmetastases thereof by moderating abnormal kinase activity comprising:administering to a patient in need thereof, an effective amount of oneor more compounds selected from the group consisting of D-chiro-inositol1,2,3,4-tetraphosphate, D-chiro-inositol 1,2,3,5-tetraphosphate,D-chiro-inositol 1,2,4,5-tetraphosphate, D-chiro-inositol1,2,4,6-tetraphosphate, D-chiro-inositol 1,2,5,6-tetraphosphate,D-chiro-inositol 1,3,4,5-tetraphosphate, D-chiro-inositol1,3,4,6-tetraphosphate, D-chiro-inositol 1,3,5,6-tetraphosphate,D-chiro-inositol 1,4,5,6-tetraphosphate, D-chiro-inositol2,3,4,5-tetraphosphate, D-chiro-inositol 2,3,4,6-tetraphosphate,D-chiro-inositol 2,3,5,6-tetraphosphate, D-chiro-inositol2,4,5,6-tetraphosphate, D-chiro-inositol 3,4,5,6-tetraphosphate,D-chiro-inositol 1,2,3,4,5-pentaphosphate, D-chiro-inositol1,2,3,4,6-pentaphosphate, D-chiro-inositol 1,2,3,5,6-pentaphosphate,D-chiro-inositol 1,2,4,5,6-pentaphosphate, D-chiro-inositol1,3,4,5,6-pentaphosphate, D-chiro-inositol2,3,4,5,6-pentaphosphate,D-chiro-inositol 1,2,3,4,5,6-hexaphosphate, D-chiro-inositol1,2,3,4,5-pentaphosphate-6-pyrophosphate, D-chiro-inositol1,2,3,4,6-pentaphosphate-5-pyrophosphate, D-chiro-inositol1,2,3,5,6-pentaphosphate-4-pyrophosphate, D-chiro-inositol1,2,4,5,6-pentaphosphate-3-pyrophosphate, D-chiro-inositol1,3,4,5,6-pentaphosphate-2-pyrophosphate, D-chiro-inositol2,3,4,5,6-pentaphosphate-1-pyrophosphate, D-chiro-inositol1,2,3-triphosphate-4,5-dipyrophosphate, D-chiro-inositol 1,2,3-triphosphate-4,6-dipyrophosphate, D-chiro-inositol1,2,3-triphosphate-5,6-dipyrophosphate, D-chiro-inositol1,2,4-triphosphate-3,5-dipyrophosphate, D-chiro-inositol1,2,4-triphosphate-3,6-dipyrophosphate, D-chiro-inositol1,2,4-triphosphate-5,6-dipyrophosphate, D-chiro-inositol1,2,5-triphosphate-3,4-dipyrophosphate, D-chiro-inositol1,2,5-triphosphate-3,6-dipyrophosphate, D-chiro-inositol1,2,5-triphosphate-4,6-dipyrophosphate, D-chiro-inositol1,2,6-triphosphate-3,4-dipyrophosphate, D-chiro-inositol1,2,6-triphosphate-3,5-dipyrophosphate, D-chiro-inositol1,2,6-triphosphate-4,6-dipyrophosphate, D-chiro-inositol1,3,4-triphosphate-2,5-dipyrophosphate, D-chiro-inositol1,3,4-triphosphate-2,6-dipyrophosphate, D-chiro-inositol1,3,4-triphosphate-5,6-dipyrophosphate, D-chiro-inositol1,3,5-triphosphate-2,4-dipyrophosphate, D-chiro-inositol1,3,5-triphosphate-2,6-dipyrophosphate, D-chiro-inositol1,3,5-triphosphate-4,6-dipyrophosphate, D-chiro-inositol1,3,6-triphosphate-2,4-dipyrophosphate, D-chiro-inositol1,3,6-triphosphate-2,5-dipyrophosphate, D-chiro-inositol1,3,6-triphosphate-4,5-dipyrophosphate, D-chiro-inositol1,4,5-triphosphate-2,3-dipyrophosphate, D-chiro-inositol1,4,5-triphosphate-2,6-dipyrophosphate, D-chiro-inositol1,4,5-triphosphate-3,6-dipyrophosphate, D-chiro-inositol1,4,6-triphosphate-2,3-dipyrophosphate, D-chiro-inositol1,4,6-triphosphate-2,5-dipyrophosphate, D-chiro-inositol1,4,6-triphosphate-3,5-dipyrophosphate, D-chiro-inositol1,5,6-triphosphate-2,3-dipyrophosphate, D-chiro-inositol1,5,6-triphosphate-2,4-dipyrophosphate, D-chiro-inositol1,5,6-triphosphate-3,4-dipyrophosphate, D-chiro-inositol2,3,4-triphosphate-1,5-dipyrophosphate, D-chiro-inositol2,3,4-triphosphate-1,6-dipyrophosphate, D-chiro-inositol2,3,4-triphosphate-5,6-dipyrophosphate, D-chiro-inositol2,3,5-triphosphate-1,4-dipyrophosphate, D-chiro-inositol2,3,5-triphosphate-1,6-dipyrophosphate, D-chiro-inositol2,3,5-triphosphate-4,6-dipyrophosphate, D-chiro-inositol2,3,6-triphosphate-1,4-dipyrophosphate, D-chiro-inositol 2,3,6-triphosphate-1,5-dipyrophosphate, D-chiro-inositol2,3,6-triphosphate-4,5-dipyrophosphate, D-chiro-inositol2,4,5-triphosphate-1,3-dipyrophosphate, D-chiro-inositol2,4,5-triphosphate-1,6-dipyrophosphate, D-chiro-inositol2,4,5-triphosphate-3,6-dipyrophosphate, D-chiro-inositol2,4,6-triphosphate-1,3-dipyrophosphate, D-chiro-inositol2,4,6-triphosphate-1,5-dipyrophosphate, D-chiro-inositol2,4,6-triphosphate-3,5-dipyrophosphate, D-chiro-inositol2,5,6-triphosphate-1,3-dipyrophosphate, D-chiro-inositol2,5,6-triphosphate-1,4-dipyrophosphate, D-chiro-inositol2,5,6-triphosphate-3,4-dipyrophosphate, D-chiro-inositol3,4,5-triphosphate-1,2-dipyrophosphate, D-chiro-inositol3,4,5-triphosphate-1,6-dipyrophosphate, D-chiro-inositol3,4,5-triphosphate-2,6-dipyrophosphate, D-chiro-inositol3,5,6-triphosphate-1,2-dipyrophosphate, D-chiro-inositol3,5,6-triphosphate-1,4-dipyrophosphate, D-chiro-inositol3,5,6-triphosphate-2,4-dipyrophosphate, D-chiro-inositol4,5,6-triphosphate-1,2-dipyrophosphate, D-chiro-inositol4,5,6-triphosphate-1,3-dipyrophosphate, D-chiro-inositol4,5,6-triphosphate-2,3-dipyrophosphate, D-chiro-inositol1-phosphate-2,3,4-tripyrophosphate, D-chiro-inositol1-phosphate-2,3,5-tripyrophosphate, D-chiro-inositol1-phosphate-2,3,6-tripyrophosphate, D-chiro-inositol1-phosphate-2,4,5-tripyrophosphate, D-chiro-inositol1-phosphate-2,4,6-tripyrophosphate, D-chiro-inositol1-phosphate-2,5,6-tripyrophosphate, D-chiro-inositol1-phosphate-3,4,5-tripyrophosphate, D-chiro-inositol1-phosphate-3,4,6-tripyrophosphate, D-chiro-inositol1-phosphate-3,5,6-tripyrophosphate, D-chiro-inositol1-phosphate-4,5,6-tripyrophosphate, D-chiro-inositol2-phosphate-1,3,4-tripyrophosphate, D-chiro-inositol2-phosphate-1,3,5-tripyrophosphate, D-chiro-inositol2-phosphate-1,3,6-tripyrophosphate, D-chiro-inositol2-phosphate-1,4,5-tripyrophosphate, D-chiro-inositol2-phosphate-1,4,6-tripyrophosphate, D-chiro-inositol2-phosphate-1,5,6-tripyrophosphate, D-chiro-inositol2-phosphate-3,4,5-tripyrophosphate, D-chiro-inositol2-phosphate-3,4,6-tripyrophosphate, D-chiro-inositol2-phosphate-3,5,6-tripyrophosphate, D-chiro-inositol2-phosphate-4,5,6-tripyrophosphate, D-chiro-inositol3-phosphate-1,2,4-tripyrophosphate, D-chiro-inositol3-phosphate-1,2,5-tripyrophosphate, D-chiro-inositol3-phosphate-1,2,6-tripyrophosphate, D-chiro-inositol3-phosphate-1,4,5-tripyrophosphate, D-chiro-inositol3-phosphate-1,4,6-tripyrophosphate, D-chiro-inositol3-phosphate-1,5,6-tripyrophosphate, and D-chiro-inositol3-phosphate-4,5,6-tripyrophosphate, wherein the one or moreD-chiro-inositol compound(s) is linked via a covalent bond to folicacid.